Defined composition gene modified t-cell products

ABSTRACT

Aspects of the invention described herein, concern approaches to make genetically modified T-cells comprising a chimeric antigen receptor for human therapy. In some alternatives, the methods utilize a selection and/or isolation of CD4+ and/or CD8+ T-cells from a mixed T-cell population, such as, peripheral blood or apheresis derived mononuclear cells. Once selected/isolated, the CD4+ and/or CD8+ T-cells are then activated, genetically modified, and propagated, preferably, in separate or isolated cultures in the presence of one or more cytokines, which support survival, engraftment and/or proliferation of the cells, as well as, preferably promoting or inducing the retention of cell surface receptors, such as CD62L, CD28, and/or CD27. Included herein are also methods of treatment, inhibition, amelioration, or elimination of a cancer by administering to a subject in need thereof, one or more types of the genetically engineered T-cells or compositions that comprise the genetically engineered T-cell prepared as described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S.Provisional Patent Application No. 61/977,751, filed Apr. 10, 2014, U.S.Provisional Patent Application No. 61/986,479, filed Apr. 30, 2014, U.S.Provisional Patent Application No. 62/058,973, filed Oct. 2, 2014, U.S.Provisional Patent Application No. 62/088,363, filed Dec. 5, 2014, U.S.Provisional Patent Application No. 62/089,730 filed Dec. 9, 2014, andU.S. Provisional Patent Application No. 62/090,845, filed Dec. 11, 2014.The entire disclosures of the aforementioned applications are expresslyincorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSCRI-090WO_SEQUENCE_LISTING.TXT, created Mar. 23, 2015, which is 2.92 kbin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Aspects of the invention concern approaches to make genetically modifiedT-cells comprising a chimeric antigen receptor. The disclosed methodsconcern the selection and/or isolation of CD4+ and/or CD8+ T-cells froma mixed T-cell population, which are then activated, geneticallymodified, and propagated in isolated cultures in the presence of one ormore cytokines, which support survival, engraftment and/or proliferationof the cells, as well as, promoting retention of cell surface receptors,such as CD62L, CD28, and/or CD27.

BACKGROUND OF THE INVENTION

Acute lymphoblastic leukemia (ALL) relapse following allogeneichematopoietic stem cell transplantation (HSCT) can occur. Accordingly,many believe such approaches are ineffective. The adoptive transfer ofhuman T lymphocytes that are engineered by gene transfer to expresschimeric antigen receptors (CARs) specific for molecules present on thesurface of tumor cells or malignant B cells has the potential toeffectively treat many advanced cancers and malignancies, as well. Inorder to be an effective and long lasting treatment, however, theadministered T-cells that comprise chimeric antigen receptors desirablyhave a high survival and proliferation rate after transfer to thepatient. The T-cells used for therapy are also desirably fit forengraftment. Despite the tremendous effort in the field, the need foradditional, effective cellular therapies remains.

SUMMARY OF THE INVENTION

Aspects of the invention described herein include methods ofmanufacturing genetically modified T-cells comprising a chimeric antigenreceptor for human therapy. Alternatives include methods utilizing aselection, enrichment, and/or isolation of CD4+ and/or CD8+ expressingT-cells, such as T-cells that are derived from thymocytes or T-cellsthat are derived from engineered precursors, desirably iPS cells, from amixed T-cell population. Once selected, enriched, or isolated, the CD4+and/or CD8+ expressing T-cells are then activated, genetically modified,and propagated, preferably, in separated, enriched, or isolated culturesin the presence of one or more cytokines, which can be providedexogenously to the T-cells, e.g., in addition to any cytokine that maybe produced by the cells or present in media and, which support,promote, induce, or contribute to survival, engraftment and/orproliferation of the cells, as well as, preferably supporting,promoting, inducing, or contributing to the retention of cell surfacereceptors, such as CD62L, CD28, and/or CD27. Included herein are alsomethods of treatment, inhibition, amelioration, or elimination of acancer by administering to a subject in need thereof, one or more typesof the genetically engineered T-cells or compositions that comprise thegenetically engineered T-cell prepared as described herein.

Some aspects of the invention described herein concern methods of makinggenetically modified T-cells, which have a chimeric antigen receptor. Bysome approaches, these methods are practiced by separating, isolating,or enriching a CD8+ expressing population of T-cells and/or a CD4+expressing population of T-cells, such as T-cells that are derived fromthymocytes or T-cells that are derived from engineered precursors,desirably iPS cells, from a mixed population of T-cells so as togenerate a separated, isolated, or enriched population of T-cells;stimulating these separated, enriched, or isolated populations ofT-cells so as to generate a stimulated population of CD8+ expressingT-cells and/or CD4+ expressing T-cells; transducing the stimulatedpopulation of CD8+ expressing T-cells and/or CD4+ expressing T-cellswith a vector, wherein the vector encodes a chimeric antigen receptorand a marker sequence, wherein said marker sequence encodes a cellsurface selectable marker, so as to generate a transduced population ofCD8+ expressing T-cells and/or CD4+ expressing T-cells; contacting thetransduced population of CD8+ expressing T-cells and/or CD4+ expressingT-cells with at least one cytokine, which can be provided exogenously tothe T-cells, e.g., in addition to any cytokine that may be produced bythe cells or present in media, so as to generate a transduced,cytokine-stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells; enriching the transduced, cytokine-stimulatedpopulation of CD8+ expressing T-cells and/or CD4+ expressing T-cells byselection of the marker sequence so as to generate a separated,enriched, or isolated population of transduced, cytokine-stimulated CD8+expressing T-cells and/or CD4+ expressing T-cells; and propagating theseparated, enriched, or isolated population of transduced,cytokine-stimulated CD8+ expressing T-cells and/or CD4+ expressingT-cells for at least two days so as to obtain said genetically modifiedT-cells, which have a chimeric antigen receptor. In some alternatives,the CD8+ expressing T-cells and/or CD4+ expressing T-cells can bepropagated for at least or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that is within arange of times defined by any two of the aforementioned time points, soas to obtain said genetically modified T-cells, which have a chimericantigen receptor. In some alternatives, the separating or enriching ofthe CD8+ population of T-cells and/or a CD4+ population of T-cells froma mixed population of T-cells is performed by affinity selection forT-cells having an epitope present on CD8 and/or CD4. In somealternatives, the separating or enriching of the CD8+ population ofT-cells and/or a CD4+ population of T-cells from a mixed population ofT-cells is performed by flow cytometry. In some alternatives, theseparating or enriching of the CD8+ population of T-cells and/or a CD4+population of T-cells from a mixed population of T-cells is performed byimmuno-magnetic selection. In some alternatives, the geneticallymodified CD8+ T-cells and/or CD4+ T-cells comprise at least one receptorthat promotes, induces, contributes to, or enhances engraftment fitness.In some alternatives, the at least one receptor is CD45 RA, CD45 RO,CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In somealternatives, the at least one receptor is CD27, CD28 and/or CD62L. Insome alternatives, the stimulating of the isolated population of T-cellsis performed by contacting the CD8+ and/or CD4+ T-cells with anantibody-bound support, such as a bead or particle. In somealternatives, the antibody-bound support comprises anti-TCR, anti-CD2,anti-CD3, anti-CD4 and/or anti-CD28 antibodies. In some alternatives,the antibody-bound support comprises anti-CD3 and/or anti-CD28antibodies. In some alternatives, the vector further comprises a firstsequence encoding a leader sequence, a second sequence encoding a ligandbinding domain, a third sequence encoding a signaling domain and afourth sequence encoding a selectable marker. In some alternatives, thevector further comprises a sequence encoding a spacer. In somealternatives, the spacer comprises an IgG4 hinge. In some alternatives,the vector is a viral vector. In some alternatives, the viral vector isderived from simian virus 40, adenoviruses, adeno-associated virus(AAV), lentivirus, or retroviruses. In some alternatives, the viralvector is a recombinant adenovirus, adeno-associated virus, lentivirusor retrovirus vector. In some alternatives, the viral vector is alentivirus vector. In some alternatives, the marker sequence encodes atruncated epidermal growth factor receptor (EGFRt). In somealternatives, the at least one cytokine comprises GM-CSF, IL-7, IL-12,IL-15, IL-18, IL-2, and/or IL-21. In some alternatives, the at least onecytokine comprises IL7, IL-15 and/or IL-21, which can be provided at 0.1ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL,0.8 ng/mL, 0.9 ng/mL, or 1.0 ng/mL or in an amount that is within arange defined by any two of the aforementioned amounts and/or at 10U/mL, 20 U/mL, 30 U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90U/mL, or 100 U/mL or in an amount that is within a range defined by anytwo of the aforementioned amounts. In some alternatives, the at leastone cytokine comprises IL-2, IL-15 and/or IL-21, wherein the amount ofcytokine is provided at 0.5 ng/mL and/or 50 U/mL. In some alternatives,the contacting is performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 days or a period of time within arange defined by any two of these values. In some alternatives, themethod is performed with isolated, purified, enriched or separated CD4+cells in the absence of, substantially depleted of, or enriched overCD8+ cells. In some alternatives, the method is performed with isolated,purified, enriched or separated CD8+ in the absence of, substantiallydepleted of, or enriched over CD4+ cells. In some alternatives, the CD4+expressing T-cells are propagated for at least 1 day, such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days orfor a period that is within a range defined by any two of theaforementioned time periods. In some alternatives, the CD8+ T-cells arepropagated at least 1 day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 days or for a period that iswithin a range defined by any two of the aforementioned time periods. Insome alternatives, the method further comprises removing theantibody-bound support, such as beads or particles. In somealternatives, the ligand binding domain of the chimeric antigen receptorcomprises an antibody, or a binding portion thereof. In somealternatives, the ligand binding domain of the chimeric antigen receptorcomprises a single chain variable fragment (scFv), or a binding portionthereof. In some alternatives, the ligand binding domain of the chimericantigen receptor comprises FMC63, or a binding portion thereof. In somealternatives, the ligand binding domain of the chimeric antigen receptoris specific for CD19. In some alternatives, the method further comprisescryopreserving the genetically modified CD8+ and/or CD4+ T-cells.

In some alternatives, the separating, enriching, or isolating of theCD8+ expressing population of T-cells and/or a CD4+ expressingpopulation of T-cells, such as T-cells that are derived from thymocytesor T-cells that are derived from engineered precursors, desirably iPScells, from a mixed population of T-cells is performed by affinityselection for T-cells having an epitope present on CD8 and/or CD4. Insome alternatives, the separating, enriching, or isolating of the CD8+expressing population of T-cells and/or a CD4+ expressing population ofT-cells from a mixed population of T-cells is performed by flowcytometry. In some alternatives, the separating, enriching, or isolatingof the CD8+ expressing population of T-cells and/or a CD4+ expressingpopulation of T-cells from a mixed population of T-cells is performed byimmuno-magnetic selection. In some alternatives, the geneticallymodified CD8+ expressing T-cells and/or CD4+ expressing T-cells compriseat least one receptor that promotes, induces, or contributes toengraftment fitness. In some alternatives, the at least one receptorthat promotes, induces, or contributes to engraftment fitness is CD45RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. Inpreferred alternatives, the at least one receptor that promotes,induces, or contributes to engraftment fitness is CD27, CD28 and/orCD62L. In some alternatives, the stimulating of the isolated, enriched,or separated population of T-cells is performed by contacting the CD8+and/or CD4+ expressing T-cells with an antibody-bound support, such as abead or particle. In some of these alternatives, the antibody-boundsupport comprises anti-TCR, anti-CD2, anti-CD3, anti-CD4 and/oranti-CD28 antibodies. In preferred alternatives, the antibody-boundsupport comprises anti-CD3 and/or anti-CD28 antibodies.

In many of the aforementioned alternatives, the vector further comprisesa first sequence encoding a leader sequence, a second sequence encodinga ligand binding domain, a third sequence encoding a signaling domainand a fourth sequence encoding a selectable marker sequence. In some ofthese alternatives, the vector further comprises a sequence encoding aspacer, which in some alternatives may comprise an IgG4 hinge. In manyof the aforementioned alternatives, the vector is a viral vector or amini-circle.

In many of the aforementioned alternatives, the viral vector is derivedfrom simian virus 40, adenoviruses, adeno-associated virus (AAV),lentivirus, or retroviruses. In some alternatives, the viral vector is arecombinant adenovirus, adeno-associated virus, lentivirus or retrovirusvector. Preferably, the viral vector is a lentivirus vector. In many ofthe aforementioned alternatives, the marker sequence encodes a truncatedepidermal growth factor receptor (EGFRt). In many of the aforementionedalternatives, the at least one cytokine that is utilized comprisesGM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2, and/or IL-21 and said cytokineis provided exogenously to the T-cells e.g., in addition to any cytokinethat may be produced by the cells or present in media.

In desirable alternatives, the at least one cytokine comprises IL-7,IL-15 and/or IL-21. In many of the aforementioned alternatives, the atleast one cytokine comprises IL-2, IL-15 and/or IL-21. In preferredalternatives, the contacting period is performed for at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days ora period of time within a range defined by any two of these time points.In many of the aforementioned alternatives, the methods are performedwith isolated, separated, or enriched populations of CD4+ expressingT-cells, such as T-cells that are derived from thymocytes or T-cellsthat are derived from engineered precursors, desirably iPS cells, in theabsence of or having a reduced amount CD8+ expressing T-cells, ascompared to a native population of unseparated, non-enriched, ornon-isolated population of T-cells. In many of the aforementionedalternatives, these methods are performed with isolated, separated, orenriched populations of CD8+ expressing T-cells, such as T-cells thatare derived from thymocytes or T-cells that are derived from engineeredprecursors, desirably iPS cells, in the absence of or having a reducedamount of CD4+ expressing T-cells, as compared to a native population ofunseparated, non-enriched, or non-isolated population of T-cells. Inmany of the aforementioned alternatives, the CD4+ expressing T-cells arepropagated for at least 1 day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that iswithin a range of times defined by any two of the aforementioned timepoints. In many of the aforementioned alternatives, the CD8+ expressingT-cells are propagated for at least 1 day, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any timethat is within a range of times defined by any two of the aforementionedtime points.

In many of the aforementioned alternatives, the methods further compriseremoving the antibody-bound support, such as beads or particles. In manyof the aforementioned alternatives, the ligand binding domain of thechimeric antigen receptor comprises an antibody, or a binding portionthereof. In many of the aforementioned alternatives, the ligand bindingdomain of the chimeric antigen receptor comprises a single chainvariable fragment (scFv), or a binding portion thereof. In many of theaforementioned alternatives, the ligand binding domain of the chimericantigen receptor comprises FMC63, or a binding portion thereof, such asis available in U.S. Pat. No. 7,446,179, herein expressly incorporatedby reference in its entirety. In many of the aforementionedalternatives, the ligand binding domain of the chimeric antigen receptoris specific for CD19. In many of the aforementioned alternatives, themethod further comprises cryopreserving the genetically modified CD8+and/or CD4+ T-cells.

Additional aspects of the invention concern a population of geneticallymodified T-cells, such as T-cells that are derived from thymocytes orT-cells that are derived from engineered precursors, comprising aplurality of affinity selected CD8+ and/or CD4+ expressing T-cells, inan enriched form, such as enriched from, in the absence of, or isolatedfrom CD8− and/or CD4− expressing T-cells, wherein said plurality ofaffinity selected CD8+ and/or CD4+ expressing T-cells have stimulatedCD2, CD3, CD4 and/or CD28 receptors, wherein said plurality of affinityselected or enriched CD8+ and/or CD4+ expressing T-cells furthercomprise a gene encoding a chimeric antigen receptor and a gene encodinga cell surface selectable marker and, wherein said plurality of affinityselected or enriched CD8+ and/or CD4+ expressing T-cells have beenre-stimulated with at least one cytokine, which can be providedexogenously to the T-cells, e.g., in addition to any cytokine that maybe produced by the cells or present in media, such as, contacting thecells with an exogenously added cytokine for at least one day, such as1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20days or any time that is within a range of times defined by any two ofthe aforementioned time points. In some alternatives, said plurality ofaffinity selected CD8+ and/or CD4+ expressing T-cells further compriseat least one receptor that promotes, induces, or contributes toengraftment fitness. In some alternatives, the at least one receptorthat promotes, induces, improves, or contributes to engraftment fitnessis CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/orCD62L. In some alternatives, the at least one receptor that promotes,induces, improves, or contributes to engraftment fitness is CD27, CD28and/or CD62L. In some alternatives, the plurality of affinity selectedCD8+ and/or CD4+ expressing T-cells further comprises a vector having afirst sequence encoding a leader sequence, a second sequence encoding aligand binding domain, a third sequence encoding a signaling domain anda fourth sequence encoding a selectable marker sequence.

Some alternatives relate to a population of genetically modifiedT-cells. In some alternatives, the population of genetically modifiedT-cells comprises a plurality of affinity selected CD8+ and/or CD4+T-cells, absence of, substantially depleted of, or enriched over CD8−and/or CD4− T-cells, wherein said plurality of affinity selected CD8+and/or CD4+ T-cells have stimulated CD2, CD3, CD4 and/or CD28 receptors,wherein said plurality of affinity selected CD8+ and/or CD4+ T-cellsfurther comprise a gene encoding a chimeric antigen receptor and a cellsurface selectable marker and, wherein said plurality of affinityselected CD8+ and/or CD4+ T-cells have been re-stimulated with at leastone cytokine. In some alternatives, said plurality of affinity selectedCD8+ and/or CD4+ T-cells further comprise at least one receptor thatpromotes, enhances, improves or contributes to engraftment fitness. Insome alternatives, the at least one receptor that promotes, enhances,improves or contributes to engraftment fitness is CD45 RA, CD45 RO,CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In somealternatives, the at least one receptor that promotes, enhances,improves or contributes to engraftment fitness is CD27, CD28 and/orCD62L. In some alternatives, the plurality of affinity selected CD8+and/or CD4+ T-cells further comprises a vector having a first sequenceencoding a leader sequence, a second sequence encoding a ligand bindingdomain, a third sequence encoding a signaling domain and a fourthsequence encoding a selectable marker. In some alternatives, the vectorfurther comprises a sequence encoding a spacer. In some alternatives,the spacer comprises an IgG4 hinge. In some alternatives, the vector isa viral vector. In some alternatives, the viral vector is derived fromsimian virus 40, adenoviruses, adeno-associated virus (AAV), lentivirus,or retroviruses. In some alternatives, the viral vector is a recombinantadenovirus, adeno-associated virus, lentivirus or retrovirus vector. Insome alternatives, the viral vector is a lentivirus vector. In somealternatives, the cell surface selectable marker encodes for a truncatedepidermal growth factor receptor (EGFRt). In some alternatives, theligand binding domain comprises an antibody, or a binding portionthereof. In some alternatives, the ligand binding domain comprises asingle chain variable fragment (scFv), or a binding portion thereof. Insome alternatives, the ligand binding domain comprises FMC63, or abinding portion thereof. In some alternatives, the ligand binding domainis specific for CD19. In some alternatives, the population comprisesisolated, purified, separated or enriched CD8+ T-cells in the absenceof, substantially depleted of, or enriched over CD4+ T-cells. In somealternatives, the population comprises isolated, purified, separated orenriched CD4+ T-cells in the absence of, substantially depleted of, orenriched over CD8+ T-cells. In some alternatives, the T cell is aprecursor T cell. In some alternatives, the precursor T cell is ahematopoietic stem cell. In some alternatives, the vector furthercomprises a sequence encoding a spacer, such as a spacer that comprisesan IgG4 hinge. In some alternatives, the vector is a viral vector. Insome alternatives, the viral vector is derived from simian virus 40,adenoviruses, adeno-associated virus (AAV), lentivirus, or retroviruses.In some alternatives, the viral vector is a recombinant adenovirus,adeno-associated virus, lentivirus or retrovirus vector. In somealternatives, the viral vector is a lentivirus vector or a mini-circle.In some alternatives, the cell surface selectable marker encodes for atruncated epidermal growth factor receptor (EGFRt). In somealternatives, the ligand binding domain comprises an antibody, or abinding portion thereof. In some alternatives, the ligand binding domaincomprises a single chain variable fragment (scFv), or a binding portionthereof. In some alternatives, the ligand binding domain comprisesFMC63, or a binding portion thereof, such as is available in U.S. Pat.No. 7,446,179, herein expressly incorporated by reference in itsentirety. In some alternatives, the ligand binding domain is specificfor CD19. In some alternatives, the population of genetically modifiedT-cells, such as T-cells that are derived from thymocytes or T-cellsthat are derived from engineered precursors, comprises isolated CD8+expressing T-cells in the absence of or having a reduced amount of CD4+expressing T-cells, as compared to a native population of unseparated,non-enriched, or non-isolated population of T-cells. In somealternatives, the population of genetically modified T-cells, such asT-cells that are derived from thymocytes or T-cells that are derivedfrom engineered precursors, comprises isolated CD4+ expressing T-cellsin the absence of or having a reduced amount of CD8+ expressing T-cells,as compared to a native population of unseparated, non-enriched, ornon-isolated population of T-cells.

Additional aspects of the invention relate to a composition or productcombination for human therapy, comprising a pharmaceutical excipient;and at least one population of the genetically modified T-cells, as setforth in the preceding paragraph. In some alternatives, the compositionor product combination comprises a population of genetically modifiedCD8+ expressing T-cells. In some alternatives, the composition orproduct combination comprises a population of genetically modified CD4+expressing T-cells. In some alternatives, the composition or productcombination comprises the population of genetically modified CD8+expressing T-cells, as set forth above, and the population ofgenetically modified CD4+ expressing T-cells, as set forth above, in amixed population or co-administered in a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1ratio or a ratio that is within a range defined by any two of theaforementioned ratios.

Additional aspects of the invention concern methods of treating,inhibiting, or ameliorating a disease in a subject in need thereofcomprising administering to the subject at least one composition orproduct combination set forth above. In some alternatives, the methodscomprise administering a composition or product combination comprisingCD4+ expressing T-cells, such as T-cells that are derived fromthymocytes or T-cells that are derived from engineered precursors, inadvance or prior to administration of the CD8+ expressing T-cells and inother alternatives, the CD8+ expressing cells, such as T-cells that arederived from thymocytes or T-cells that are derived from engineeredprecursors, are administered before the CD4+ expressing T-cells. In manyalternatives, the subject is identified or selected to receive ananti-cancer therapy. In many of the aforementioned methods, the approachalso involves measuring or evaluating an inhibition of a disease. Inmany of the aforementioned methods, the approach also involves providingsaid subject an additional anti-cancer therapy before, during, or afteradministration of a composition or product combination set forth above.

In many of the aforementioned methods, a composition or productcombination is administered to said subject by adoptive cell transfer.In some alternatives, a composition or product combination areadministered to said subject after said subject has received anotherform of anti-cancer therapy. In many of the aforementioned methods, thesubject is suffering from leukemia. In many of the aforementionedmethods, the subject has recurrent and/or chemotherapy refractory CD19+childhood acute lymphoblastic leukemia (ALL). In many of theaforementioned methods, the subject has recurrent and/or chemotherapyrefractory CD19+ acute lymphoblastic leukemia (ALL). In many of theaforementioned methods, the subject is suffering from an autoimmunedisease. In many of the aforementioned methods, the subject is sufferingfrom a post-HSCT relapse.

Accordingly, some aspects of the present invention relate to thefollowing alternatives:

1. A method of making genetically modified T-cells, which have achimeric antigen receptor, comprising:

separating or enriching a CD8+ expressing population of T-cells and/or aCD4+ expressing population of T-cells, such as T-cells that are derivedfrom thymocytes or T-cells that are derived from engineered precursors,desirably iPS cells, from a mixed population of T-cells so as togenerate a separated or enriched population of T-cells;

stimulating the separated or enriched population of T-cells so as togenerate a stimulated population of CD8+ T-cells and/or CD4+ T-cells;

transducing the stimulated population of CD8+ T-cells and/or CD4+T-cells with a vector, wherein the vector encodes a chimeric antigenreceptor and a marker sequence, wherein said marker sequence encodes acell surface selectable marker, so as to generate a transducedpopulation of CD8+ T-cells and/or CD4+ T-cells;

contacting the transduced population of CD8+ T-cells and/or CD4+ T-cellswith at least one cytokine, which can be provided exogenously to theT-cells, e.g., in addition to any cytokine that may be produced by thecells or present in media, such as for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or for a period that iswithin a range defined by any two of the aforementioned time periods, soas to generate a transduced, cytokine-stimulated population of CD8+T-cells and/or CD4+ T-cells;

enriching the transduced, cytokine-stimulated population of CD8+ T-cellsand/or CD4+ T-cells by selection of the marker sequence so as togenerate an enriched population of transduced, cytokine-stimulated CD8+T-cells and/or CD4+ T-cells; and

propagating the enriched population of transduced, cytokine-stimulatedCD8+ T-cells and/or CD4+ T-cells for at least one day, such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days orfor a period that is within a range defined by any two of theaforementioned time periods, so as to obtain said genetically modifiedT-cells, which have a chimeric antigen receptor.

2. The method of alternative 1, wherein the separating or enriching ofthe CD8+ population of T-cells and/or a CD4+ population of T-cells froma mixed population of T-cells is performed by affinity selection forT-cells having an epitope present on CD8 and/or CD4.

3. The method of alternative 1 or 2, wherein the separating or enrichingof the CD8+ population of T-cells and/or a CD4+ population of T-cellsfrom a mixed population of T-cells is performed by flow cytometry.

4. The method of alternative 1 or 2, wherein the separating or enrichingof the CD8+ population of T-cells and/or a CD4+ population of T-cellsfrom a mixed population of T-cells is performed by immuno-magneticselection.

5. The method of any one of alternatives 1-4, wherein the geneticallymodified CD8+ T-cells and/or CD4+ T-cells comprise at least one receptorthat promotes, induces, contributes to, or enhances engraftment fitness.

6. The method of alternative 5, wherein the at least one receptor isCD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/orCD62L.

7. The method of alternative 5 or 6, wherein the at least one receptoris CD27, CD28 and/or CD62L.

8. The method of any one of alternatives 1-7, wherein the stimulating ofthe isolated population of T-cells is performed by contacting the CD8+and/or CD4+ T-cells with an antibody-bound support, such as a bead orparticle.

9. The method of alternative 8, wherein the antibody-bound supportcomprises anti-TCR, anti-CD2, anti-CD3, anti-CD4 and/or anti-CD28antibodies.

10. The method of alternative 8 or 9, wherein the antibody-bound supportcomprises anti-CD3 and/or anti-CD28 antibodies.

11. The method of any one of alternatives 1-10, wherein the vectorfurther comprises a first sequence encoding a leader sequence, a secondsequence encoding a ligand binding domain, a third sequence encoding asignaling domain and a fourth sequence encoding a selectable marker.

12. The method of any one of alternatives 1-11, wherein the vectorfurther comprises a sequence encoding a spacer.

13. The method of alternative 12, wherein the spacer comprises an IgG4hinge.

14. The method of any one of alternatives 1-13, wherein the vector is aviral vector.

15. The method of alternative 14, wherein the viral vector is derivedfrom simian virus 40, adenoviruses, adeno-associated virus (AAV),lentivirus, or retroviruses.

16. The method of alternative 14 or 15, wherein the viral vector is arecombinant adenovirus, adeno-associated virus, lentivirus or retrovirusvector.

17. The method of any one of alternatives 14-16, wherein the viralvector is a lentivirus vector.

18. The method of any one of alternatives 1-17, wherein the markersequence encodes a truncated epidermal growth factor receptor (EGFRt).

19. The method of any one of alternatives 1-18, wherein the at least onecytokine comprises GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2 and/or IL-21.

20. The method of any one of alternatives 1-19, wherein the at least onecytokine comprises IL7, IL-15 and/or IL-21, which can be provided at 0.1ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL,0.8 ng/mL, 0.9 ng/mL, or 1.0 ng/mL or in an amount that is within arange defined by any two of the aforementioned amounts and/or at 10U/mL, 20 U/mL, 30 U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90U/mL, or 100 U/mL or in an amount that is within a range defined by anytwo of the aforementioned amounts.

21. The method of any one of alternatives 1-19, wherein the at least onecytokine comprises IL-2, IL-15 and/or IL-21, wherein the amount ofcytokine is provided at 0.5 ng/mL and/or 50 U/mL.

22. The method of any one of alternatives 1-21, wherein the contactingis performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 days or a period of time within a range defined by anytwo of these values.

23. The method of any one of alternatives 1-20 or 22, wherein the methodis performed with isolated, purified, enriched or separated CD4+ cellsin the absence of, substantially depleted of, or enriched over CD8+cells.

24. The method of any one of alternatives 1-19, 21 or 22, wherein themethod is performed with isolated, purified, enriched or separated CD8+in the absence of, substantially depleted of, or enriched over CD4+cells.

25. The method of any one of alternatives 1-20, 22 or 23, wherein theCD4+ expressing T-cells are propagated for at least 1 day, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 daysor for a period that is within a range defined by any two of theaforementioned time periods.

26. The method of any one of alternatives 1-19, 21, 22 or 24, whereinthe CD8+ T-cells are propagated at least 1 day, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or for aperiod that is within a range defined by any two of the aforementionedtime periods.

27. The method of any one of alternatives 8-26, wherein the methodfurther comprises removing the antibody-bound support, such as beads orparticles.

28. The method of any one of alternatives 1-27, wherein the ligandbinding domain of the chimeric antigen receptor comprises an antibody,or a binding portion thereof.

29. The method of any one of alternatives 1-28, wherein the ligandbinding domain of the chimeric antigen receptor comprises a single chainvariable fragment (scFv), or a binding portion thereof.

30. The method of any one of alternatives 1-29, wherein the ligandbinding domain of the chimeric antigen receptor comprises FMC63, or abinding portion thereof.

31. The method of any one of alternatives 1-30, wherein the ligandbinding domain of the chimeric antigen receptor is specific for CD19.

32. The method of any one of alternatives 1-31, wherein the methodfurther comprises cryopreserving the genetically modified CD8+ and/orCD4+ T-cells.

33. A population of genetically modified T-cells comprising:

-   -   a plurality of affinity selected CD8+ and/or CD4+ T-cells,        absence of, substantially depleted of, or enriched over CD8−        and/or CD4− T-cells, wherein said plurality of affinity selected        CD8+ and/or CD4+ T-cells have stimulated CD2, CD3, CD4 and/or        CD28 receptors, wherein said plurality of affinity selected CD8+        and/or CD4+ T-cells further comprise a gene encoding a chimeric        antigen receptor and a cell surface selectable marker and,        wherein said plurality of affinity selected CD8+ and/or CD4+        T-cells have been re-stimulated with at least one cytokine.

34. The population of genetically modified T-cells of alternative 33,wherein said plurality of affinity selected CD8+ and/or CD4+ T-cellsfurther comprise at least one receptor that promotes, enhances, improvesor contributes to engraftment fitness.

35. The population of genetically modified T-cells of alternative 33 or34, wherein the at least one receptor that promotes, enhances, improvesor contributes to engraftment fitness is CD45 RA, CD45 RO, CCR7, CD25,CD127, CD57, CD137, CD27, CD28 and/or CD62L.

36. The population of genetically modified T-cells of alternative 34 or35, wherein the at least one receptor that promotes, enhances, improvesor contributes to engraftment fitness is CD27, CD28 and/or CD62L.

37. The population of genetically modified T-cells of any one ofalternatives 33-36, wherein the plurality of affinity selected CD8+and/or CD4+ T-cells further comprises a vector having a first sequenceencoding a leader sequence, a second sequence encoding a ligand bindingdomain, a third sequence encoding a signaling domain and a fourthsequence encoding a selectable marker.

38. The population of genetically modified T-cells of alternative 37,wherein the vector further comprises a sequence encoding a spacer.

39. The population of genetically modified T-cells of alternative 38,wherein the spacer comprises an IgG4 hinge.

40. The population of genetically modified T-cells of any one ofalternatives 33-39, wherein the vector is a viral vector.

41. The population of genetically modified T-cells of alternative 40,wherein the viral vector is derived from simian virus 40, adenoviruses,adeno-associated virus (AAV), lentivirus, or retroviruses.

42. The population of genetically modified T-cells of alternative 40 or41, wherein the viral vector is a recombinant adenovirus,adeno-associated virus, lentivirus or retrovirus vector.

43. The population of genetically modified T-cells of any one ofalternatives 40-42, wherein the viral vector is a lentivirus vector.

44. The population of genetically modified T-cells of any one ofalternatives 33-43, wherein the cell surface selectable marker encodesfor a truncated epidermal growth factor receptor (EGFRt).

45. The population of genetically modified T-cells of any one ofalternatives 37-44, wherein the ligand binding domain comprises anantibody, or a binding portion thereof.

46. The population of genetically modified T-cells of any one ofalternatives 37-45, wherein the ligand binding domain comprises a singlechain variable fragment (scFv), or a binding portion thereof.

47. The population of genetically modified T-cells of any one ofalternatives 37-46, wherein the ligand binding domain comprises FMC63,or a binding portion thereof.

48. The population of genetically modified T-cells of any one ofalternatives 37-47, wherein the ligand binding domain is specific forCD19.

49. The population of genetically modified T-cells of any one ofalternatives 33-48, wherein the population comprises isolated, purified,separated or enriched CD8+ T-cells in the absence of, substantiallydepleted of, or enriched over CD4+ T-cells.

50. The population of genetically modified T-cells of any one ofalternatives 33-48, wherein the population comprises isolated, purified,separated or enriched CD4+ T-cells in the absence of, substantiallydepleted of, or enriched over CD8+ T-cells.

51. A composition or product combination for human therapy, comprising:

-   -   a pharmaceutical excipient; and    -   at least one population of genetically modified T-cells        according to any one or more of alternatives 33-50.

52. The composition or product combination of alternative 51, whereinthe composition or product combination comprises the population ofgenetically modified T-cells of alternative 49.

53. The composition or product combination of alternative 47, whereinthe composition or product combination comprises the population ofgenetically modified T-cells of alternative 50.

54. The composition or product combination of alternative 47, whereinthe composition or product combination comprises the population ofgenetically modified T-cells of alternative 49 and the population ofgenetically modified T-cells of alternative 50, mixed or co-administeredin a 1:1 ratio.

55. A method of treating, inhibiting, or ameliorating a disease in asubject in need thereof comprising:

administering to the subject at least one composition or productcombination of any one or more of alternatives 51-54.

56. The method of alternative 55, wherein the method comprisesadministering the composition or product combination of alternative 52.

57. The method of alternative 55, wherein the method comprisesadministering the composition or product combination of alternative 53.

58. The method of alternative 56, wherein the method further comprisesadministering the composition or product combination of alternative 53.

59. The method of alternative 57, wherein the method further comprisesadministering the composition or product combination of alternative 52.

60. The method of alternative 55, wherein the method comprisesadministering the composition or product combination of alternative 54,such as by an approach, wherein the CD8+ expressing T-cells areadministered in advance of the CD4+ expressing T-cells, such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 minutes before the CD4+T-cells are administered or a time period that is within a range definedby any two of the aforementioned times.

61. The method of any one or more of alternatives 55-60, wherein thesubject is identified or selected to receive an anti-cancer therapy.

62. The method of any one or more of alternatives 55-61, furthercomprising measuring or evaluating an inhibition of a disease.

63. The method of any one or more of alternatives 55-62, furthercomprising providing said subject an additional anti-cancer therapybefore, during, or after administration of the composition or productcombination of any one or more of alternatives 51-54.

64. The method of any one or more of alternatives 55-63, wherein thecomposition or product combination of any one or more of alternatives51-54 are administered to said subject by adoptive cell transfer.

65. The method of any one or more of alternatives 55-64, wherein thecomposition or product combination of any one or more of alternatives51-54 are administered to said subject after said subject has receivedanother form of anti-cancer therapy.

66. The method of any one or more of alternatives 55-65, wherein thecomposition or product combination of any one or more of alternatives51-54 are administered to said subject after said subject has receivedanother form of anti-cancer therapy.

67. The method of any one or more of alternatives 55-66, wherein thesubject is suffering from leukemia.

68. The method of any one or more of alternatives 55-67, wherein thesubject has recurrent and/or chemotherapy refractory CD19+ childhoodacute lymphoblastic leukemia (ALL).

69. The method of any one or more of alternatives 55-68, wherein thesubject has recurrent and/or chemotherapy refractory CD19+ acutelymphoblastic leukemia (ALL).

70. The method of any one or more of alternatives 55-69, wherein thesubject is suffering from an autoimmune disease.

71. The method of any one or more of alternatives 55-70, wherein thesubject is suffering from a post-HSCT relapse.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the absolute number of anti-CD19 CAR bearing T-cells in 11patients following adoptive transfer of the CAR bearing T-cells from Day0 to Day 65.

FIG. 1B shows the absolute number of anti-CD19 CAR bearing T-cellsfollowing three doses of treatment.

FIG. 2A shows the persistence of anti-CD19 CAR bearing T-cells inperipheral blood of patients following adoptive transfer of the CARbearing T-cells from Day 0 to Day 65. FIG. 2B shows the persistence ofanti-CD19 car bearing T-cells following three doses of treatment.

FIG. 3A shows the persistence of anti-CD19 CAR bearing T-cells in bonemarrow of patients following adoptive transfer of the CAR bearingT-cells from Day 0 to Day 65. FIG. 3B shows the persistence of anti-CD19CAR bearing T-cells in bone marrow patients following three doses oftreatment.

FIG. 4 shows the percent acute lymphoblastic leukemia (ALL) in patient Xfollowing three doses.

FIG. 5 shows a table of the patient characteristics that were used inthe studies.

FIG. 6 shows the development of CNS lymphoma, as related to disease anddosage. As shown, two bar graphs indicate the number of patientssubjected to the treatment comprising anti-CD19 CAR bearing T-cells andtheir severity of disease following 3 treatments.

FIG. 7 shows grade 4 encephalopathy in an abnormal MRI of a patientsuffering from ALL. The first panel indicates the results followingtreatment using anti-CD19 CAR bearing T-cells.

FIG. 8 shows acute Skin Graft-versus-host disease (GVHD) followingtreatment with anti-CD19 CAR bearing T-cells. As shown in panel A, S03developed de novo grade 2 acute skin GVHD d17 post CAR T-cellengraftment. In panel B, a skin biopsy revealed that only 9% of skinlocalized T-cells marked EGFRt+ while 79% of circulating T-cells markedEGFRt+. In panel C, peripheral blood at the same time showed themajority of the T-cells were CAR+. This subject was treated with a 2week course of 1 mg/kg of prednisone, followed by a rapid taper over asix week period and resolution of the GVHD. Despite the prednisone, thesubject has ongoing persistent CAR+ T-cells.

FIG. 9 shows a table of patients experiencing Ig or TCR as a marker ofminimal residual disease (MRD) following treatment using anti-CD19 CARbearing T-cells.

FIG. 10 shows a table of patient profiles after allogeneic hematopoieticstem cell transplantation of anti-CD19CAR T-cells.

FIG. 11 shows the FACS scatter analysis of the cells isolated from theperipheral blood and CSF following hematopoietic stem celltransplantation (HSCT) of anti-CD19 CAR T-cells.

FIG. 12 shows the tumor burden vs the response after three doses ofhematopoietic stem cell transplantation of anti-CD19 CAR T-cells.

FIG. 13 shows the remission duration of patients following three dosesof hematopoietic stem cell transplantation.

FIG. 14 shows the tumor burden vs the response after two doses ofhematopoietic stem cell transplantation of anti-CD19CAR T-cells at day7.

FIG. 15 shows the magnitude and duration of CAR/EGFRt+ T-cellpersistence in a patient after three doses of hematopoietic stem celltransplantation of anti-CD19 CAR T-cells.

FIG. 16 shows the duration of B cell aplasia after three doses ofhematopoietic stem cell transplantation of anti-CD19 CAR T-cells.

FIG. 17 shows the CRS profile of a patient after three doses ofhematopoietic stem cell transplantation of anti-CD19 CAR T-cells.

FIG. 18 shows two flow diagrams illustrating the manufacturing ofgenetically modified T-cells. As depicted, for the initial expansionmethodology bulk processing was not always initiated immediately. Aportion of the apheresis product was always taken for Ficoll processing.If no bulk cultures were to be initiated then that product separated byFicoll was cryopreserved.

FIG. 19 shows a flow diagram of comparisons of different methods ofmaking genetically modified T-cells that have chimeric antigenreceptors.

FIG. 20 shows the initial comparisons in cell growth using differentconcentrations of cells in the starter culture. As shown, PD0064 are thePD0063 donor which are enriched CD8+ expressing T-cells were used totest starting cell density in T25 flask on initiation. The experimentstested two cell densities and both showed greater viability andexpansion when the volume was increased early during the experiment.

FIG. 21 shows an initiation comparison of cell growth. This was the fullscale data used to transition to the update manufacturing processcurrently being used.

FIG. 22 shows a flow diagram illustrating the variations of cocktails ofcytokines that were used to test CD4+ and CD8+ expressing T-cells forgrowth.

FIGS. 23A-F illustrate the comparison of cytokines that were testedduring the growth of CD4+ and CD8+ expressing T-cells.

FIGS. 24A-F illustrate the comparison of cytokines that were testedduring the growth of CD4+ and CD8+ expressing T-cells.

FIGS. 25A-B shows a cytokine comparisons for cell growth. For PD0059donor sample the same cytokine testing experiment was performed as inPD0051 except testing a new CD4 and CD8 donor (PD0057).

FIG. 26 shows a flow diagram illustrating the initial expansionmethodology and the current expansion methodology derived from theexperimentation of testing different cytokine mixtures.

FIG. 27 shows expansion comparisons of samples PD0080, PD084 and PD0085.These experiments were repeats of the “early expansion” methodology.

FIG. 28 shows the tests on PD0044 for producing the genetically modifiedcells. For the first scale up PLAT-02 (Phase I and Phase II trial)“pre-qual” from cryopreserved selected cells were used.

FIG. 29 shows the tests on PD0046 for producing the genetically modifiedcells from the scale up of PLAT-02 “Qual run #1”.

FIG. 30 shows the tests on PD0063 for producing the genetically modifiedcells. From the scale up or PLAT-02 “Qual run #2”. Growth curves showTNC from both V-197 bags of cells together. Bead removal and EGFRtenrichment occurred afterward. D+14 for CD4+ expressing T-cells and D+15for CD8+ expressing T-cells. CD8+ expressing T-cells grown in IL-2 (50U/mL)/IL-15 (0.5 ng/mL), CD4+ expressing T-cells grown in IL-7 (5ng/mL)/IL-15 (0.5 ng/mL). Bead removal and EGFRt enrichment occurred atD12 for CD4+ expressing T-cells and D13 for CD8+ expressing T-cells.

FIGS. 31A-B show the expansion of cells from bulk PBMC cultures whengrown in the presence of cytokines. As shown, CD4+ expressing T-cells() were grown in the presence of IL-2 and IL-15. CD8+ expressingT-cells (▪) were grown in the presence of IL-7 and IL-15.

FIGS. 32A-B show the expansion of enriched CD8+ and CD4+ expressingT-cells in cytokine mixtures. As shown in sample PD0044, enriched CD8+expressing T-cells were expanded in the presence of IL-2 and IL-15. Insample PD0044, enriched CD4+ expressing T-cells were expanded in thepresence of IL-7 and IL-15 for over 20 days.

FIG. 33 shows expansion of enriched CD4+ and CD8+ expressing T-cellsusing an earlier methodology as shown in the flow chart of FIG. 18.Shown are experiments of cells in sample 14602-S01, 14602-S02, and14602-S03/14602-S03-02.

FIG. 34 shows expansion of enriched CD4+ and CD8+ expressing T-cellsusing an earlier methodology as shown in the flow chart of FIG. 18.Shown are experiments of cells in sample 14602-S04/14602-S04-02,14602-S05, 14602-S06 and 14602-S06-2/14602-S06-04.

FIG. 35 shows the expansion of enriched CD4+ and CD8+ expressing T-cellsusing the “Early Expansion methodology as shown in the flow chart ofFIG. 18. Shown are experiments of cells in sample 14602-S07, 14602-S08,and 14602-S09.

FIG. 36 shows the expansion of cells in samples 14602-S10, 14602-S11,14602-S12, and 14602-S13.

FIG. 37 shows the expansions of cells in samples 14602-S14, 14602-S15,and 14602-S16.

FIG. 38 shows the enriched CD4+ and CD8+ expressing T-cells extendedphenotypes after cell expansion.

FIGS. 39A-B shows the survival of a mouse injected with cells from thesample PD00044 and PD00046. PD00046 cells were noted for havingexpression of engraftment fitness markers (CD27, CD28, CD127, andCD62L).

FIG. 40 shows the average tumor progression in mice treated with cellsfrom the PD0044 and PD0046 cell batches.

FIG. 41 shows the experimental set up of testing tumor progression inmice treated with T-cells that are manufactured for engraftment fitness.

FIG. 42 shows a comparison between CD4+ and CD8+ expressing T-cells fromthe samples PD0051 and PD00055 on the day of administration into animalsand the cytokine growth conditions.

FIG. 43, shows a comparison of the three groups of mice that weretreated with PBS, PD0051 (normal expansion cells) and PD00055 (cellsexpanded in the presence of cytokine combinations)

FIG. 44, shows the T-cell persistence in peripheral blood of the mice bydetection of the EGFRt CAR marker.

FIG. 45 shows the experimental set up for testing three groups of micein order to determine an in vivo difference in killing ability betweencells that have been grown in various cytokine conditions in vitro withrepeat antigen encounters.

FIG. 46 shows the tumor progression of PD0051 and PD0055 treated micefollowing treatment from day 0 to day 120.

FIG. 47 shows PD0051 and PD0055 cells challenged with repeat antigenexposure.

FIG. 48 shows JME13-29 repeated Raji Tumor challenge of “normalexpansion cells” versus T-cells pulsed with cytokine combinations.

FIG. 49 shows the experimental set up to determine if there is an invivo difference in killing ability between cells that have been grownunder the same conditions as PLAT-01 (Phase I clinical trial) andPLAT-02 (Phase I and Phase II clinical trial), as well as, “in between”protocols.

FIG. 50 shows a table that indicates the cell products and the levels ofEGFRt from the CD8+ and CD4+ expressing T-cells.

FIG. 51 shows the average tumor progression after treatment with T-cellsat specific dose titrations.

FIG. 52 shows the PLAT comparisons within dosing groups of T-celltreatments.

FIGS. 53A-E show the comparison of T-cells from the products ofPLAT-1.00, PLAT-1.33, PLAT-1.67 and PLAT-2.00 at similar dosingconcentrations. The figure illustrates the tumor progression ofindividual animals. The start of each row shows the grouped for eachPLAT group with the various dose titrations, followed by the individualgroups teased out.

FIG. 54 shows the dose titration survival of mice treated with T-cellsfrom the products of PLAT-1.00, PLAT-1.33, PLAT-1.67 and PLAT-2.00. Thefigure illustrates the PLAT comparison within dosing groups of celltreatments.

FIG. 55 shows the PLAT comparison of survival curves. The figureillustrates the average tumor progression after treatment with T-cellsat specific doses.

FIG. 56 shows the survival of the mice in response to T-cell treatmentin which the T-cells were grown in normal expansion methods or grown inthe presence of cytokines. The figure also shows if the mice developedxenoGraft Host disease after T-cell treatment

FIG. 57 shows the survival of the mice in response to T-cell treatmentin which the T-cells were grown in normal expansion methods or grown inthe presence of cytokines. The figure also shows if the mice developedxenoGraft Host disease after T-cell treatment

FIGS. 58A-C show the survival of the mice in response to T-celltreatment in which the T-cells were grown in normal expansion methods orgrown in the presence of cytokines. The figures also show if the micedeveloped xenoGraft Host disease after T-cell treatment

FIGS. 59A-H show the survival of the mice in response to T-celltreatment in which the T-cells were grown in normal expansion methods orgrown in the presence of cytokines. The figures also show if the micedeveloped xenoGraft Host disease after T-cell treatment

FIGS. 60A-D show the survival of the mice in response to T-celltreatment in which the T-cells were grown in normal expansion methods orgrown in the presence of cytokines. The figures also show if the micedeveloped xenoGraft Host disease after T-cell treatment.

FIG. 61 shows a table of JME14-03 Group N, at Day 51 post-T celltreatment at 51 days and at 90 days.

DETAILED DESCRIPTION

The following definitions are provided to facilitate understanding ofseveral of the alternatives described herein.

As used herein, “a” or “an” can mean one or more than one.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA)or ribonucleic acid (RNA), oligonucleotides, fragments generated by thepolymerase chain reaction (PCR), and fragments generated by any ofligation, scission, endonuclease action, exonuclease action, and bysynthetic generation. Nucleic acid molecules can be composed of monomersthat are naturally-occurring nucleotides (such as DNA and RNA), oranalogs of naturally-occurring nucleotides (e.g., enantiomeric forms ofnaturally-occurring nucleotides), or a combination of both. Modifiednucleotides can have alterations in sugar moieties and/or in pyrimidineor purine base moieties. Sugar modifications include, for example,replacement of one or more hydroxyl groups with halogens, alkyl groups,amines, and azido groups, or sugars can be functionalized as ethers oresters. Moreover, the entire sugar moiety can be replaced withsterically and electronically similar structures, such as aza-sugars andcarbocyclic sugar analogs. Examples of modifications in a base moietyinclude alkylated purines and pyrimidines, acylated purines orpyrimidines, or other well-known heterocyclic substitutes. Nucleic acidmonomers can be linked by phosphodiester bonds or analogs of suchlinkages. Analogs of phosphodiester linkages include phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.The term “nucleic acid molecule” also includes so-called “peptidenucleic acids,” which comprise naturally-occurring or modified nucleicacid bases attached to a polyamide backbone. Nucleic acids can be eithersingle stranded or double stranded.

“Genetically modify,” as used herein, refers to a process for modifyingan organism or a cell such as a bacterium, T-cell, bacterial cell,eukaryotic cell, insect, plant or mammal with genetic material, such asnucleic acid, that has been altered using genetic engineeringtechniques. For example, nucleic acid such as DNA can be inserted in thehost genome by first isolating and copying the genetic material ofinterest using molecular cloning methods to generate a DNA sequence, orby synthesizing the DNA, and then inserting this construct into the hostorganism. Genes can also be removed, or “knocked out”, using a nuclease.Gene targeting is a different technique that uses homologousrecombination to change an endogenous gene, and can be used to delete agene, remove exons, add a gene, or introduce point mutations.

Genetic modification performed by transduction is described herein.“Transduction” refers to methods of transferring genetic material, suchas, for example, DNA or RNA, to a cell by way of a vector. Commontechniques use viral vectors, electroporation, and chemical reagents toincrease cell permeability. The DNA can be transferred by a virus, orvia a viral vector. As described herein, methods are provided formodifying immune CD4+ and/or CD8+ T-cells. In order to achieve highexpression of therapeutic genes and/or increase the amount of chimericantigen receptors on a cell surface, for example, T-cells can betransduced with genetic material encoding a chimeric antigen receptor.T-cells can be genetically modified using a virus. Viruses commonly usedfor gene therapy are adenovirus, adeno-associated virus (AAV),retroviruses and lentiviruses.

Various transduction techniques have been developed, which utilizerecombinant infectious virus particles for delivery of the nucleic acidencoding a chimeric antigen receptor. This represents a currentlypreferred approach to the transduction of T lymphocytes. As describedherein, the viral vectors used for transduction can include virusvectors derived from simian virus 40, adenoviruses, adeno-associatedvirus (AAV), lentiviral vectors, and retroviruses. Thus, gene transferand expression methods are numerous but essentially function tointroduce and express genetic material in mammalian cells. Several ofthe above techniques can be used to transduce hematopoietic or lymphoidcells, including calcium phosphate transfection, protoplast fusion,electroporation, and infection with recombinant adenovirus,adeno-associated virus, lentivirus, or retrovirus vectors. Primary Tlymphocytes have been successfully transduced by electroporation and byretroviral or lentiviral infection. As such, retroviral and lentiviralvectors can provide a highly efficient method for gene transfer ineukaryotic cells. Retroviral and lentiviral vectors provide highlyefficient methods for gene transfer into T-cells. Moreover, retroviralor lentiviral integration takes place in a controlled fashion andresults in the stable integration of one or a few copies of the newgenetic information per cell.

An “expression vector” or a vector, as described herein, is a nucleicacid molecule encoding a gene that is expressed in a host-cell.Typically, an expression vector comprises a transcription promoter, agene, and a transcription terminator. Gene expression is usually placedunder the control of a promoter, and such a gene is said to be “operablylinked to” the promoter. Similarly, a regulatory element and a corepromoter are operably linked if the regulatory element modulates theactivity of the core promoter.

In some alternatives, a method of making genetically modified T-cells,such as T-cells that are derived from thymocytes or T-cells that arederived from engineered precursors, which have a chimeric antigenreceptor is provided, wherein the method comprises separating,isolating, or enriching a CD8+ expressing population of T-cells and/or aCD4+ expressing population of T-cells from a mixed population of T-cellsso as to generate an isolated, separated, or enriched population ofT-cells, stimulating the isolated, separated or enriched population ofT-cells so as to generate a stimulated population of CD8+ expressingT-cells and/or CD4+ expressing T-cells, transducing the stimulatedpopulation of CD8+ expressing T-cells and/or CD4+ expressing T-cellswith a vector, wherein the vector encodes a chimeric antigen receptorand a marker sequence, wherein said marker sequence encodes a cellsurface selectable marker, so as to generate a transduced population ofCD8+ expressing T-cells and/or CD4+ expressing T-cells, contacting thetransduced population of CD8+ expressing T-cells and/or CD4+ expressingT-cells, for at least one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time that iswithin a range of times defined by any two of the aforementioned timepoints, with at least one cytokine so as to generate a transduced,cytokine-stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells, enriching, separating, or isolating the transduced,cytokine-stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells by selection of the marker encoded by the markersequence so as to generate an enriched, separated or isolated populationof transduced, cytokine-stimulated CD8+ expressing T-cells and/or CD4+expressing T-cells and propagating the enriched, separated, or isolatedpopulation of transduced, cytokine-stimulated CD8+ expressing T-cellsand/or CD4+ expressing T-cells for at least one day, such as 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or anytime that is within a range of times defined by any two of theaforementioned time points, so as to obtain said genetically modifiedT-cells, which have a chimeric antigen receptor. In some alternatives ofthe method, the vector is a viral vector. In some alternatives of themethod, the viral vector is derived from simian virus 40, adenoviruses,adeno-associated virus (AAV), lentivirus, or retroviruses. In somealternatives of the method, the viral vector is a recombinantadenovirus, adeno-associated virus, lentivirus or retrovirus vector. Insome alternatives of the method, the viral vector is a lentivirusvector.

A “leader sequence” also known as the “5′ untranslated region (5′ UTR),is the region of mRNA that is located upstream from the initiation codonand is important in regulating the translation of an mRNA transcript. Insome alternatives of the method of making genetically modified T-cells,which have a chimeric antigen receptor, the vector encoding the chimericantigen receptor comprises a sequence encoding a leader sequence.

A “ligand” as described herein refers to a small molecule that can forma complex with another molecule or biomolecule for a biological purposesuch as, for example, signal triggering. Binding can occur throughintermolecular forces, for example ionic bonds, hydrogen bonds, and vander walls interactions. Ligand binding to a receptor protein can alterthe three dimensional structure and determine its functional state.

By way of example and not of limitation, ligands can include substrates,proteins, small molecules, inhibitors, activators and neurotransmitters.The strength of binding of a ligand is referred to as the bindingaffinity and can be determined by direct interactions and solventeffects. A ligand can be bound by a “ligand binding domain.” A ligandbinding domain can refer to a conserved sequence in a structure that canbind a specific ligand. Without being limiting, a ligand binding domaincan be a specific protein domain that is specific for a ligand orligands.

Specific” or “Specificity” can refer to the characteristic of a ligandfor the binding partner or alternatively, the binding partner for theligand, and can include complementary shape, charge and hydrophobicspecificity for binding. Specificity for binding can includestereospecificity, regioselectivity and chemoselectivity.

A “signaling domain,” also known as a “Co-stimulatory domain” is anintracellular or cytoplasmic domain of a protein or a receptor proteinthat interacts with the interior of the cells and functions by relayinga signal. The portion of the protein that resides in the intracellularportion of the cell is also referred to as the “endodomain.” Thisinteraction can occur through the intracellular domain communicating viaspecific protein-protein or protein-ligand interactions with an effectormolecule or an effector protein, which in turn can send the signal alonga signal chain to its destination.

The signaling or co-stimulatory domain also refers to a signaling moietythat provides to T-cells a signal which, in addition to the primarysignal provided by for instance the CD3 zeta chain of the TCR/CD3complex, mediates a T-cell response, such as, for example, an immuneresponse, activation, proliferation, differentiation, cytokinesecretion, cytolytic activity, perforin and/or granzyme activity and thelike. A signaling or co-stimulatory domain can include all or a portionof, but is not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and/or a ligand that specifically binds with CD83.

“Selectable marker sequence,” is a gene introduced into a vector or acell that confers a trait for artificial selection. A selectable markersequence or marker sequence can be a screenable marker to allow aresearcher to distinguish between wanted and unwanted cells, or toenrich for a specific cell type. In some alternatives of the method ofmaking genetically modified T-cells, which have a chimeric antigenreceptor, a vector is provided wherein the vector encodes a chimericantigen receptor comprising a marker sequence, wherein said markersequence encodes a cell surface selectable marker.

In some alternatives, a method of making genetically modified T-cells,which have a chimeric antigen receptor is provided, wherein the methodcomprises separating, isolating or enriching a CD8+ expressingpopulation of T-cells and/or a CD4+ expressing population of T-cells,such as T-cells that are derived from thymocytes or T-cells that arederived from engineered precursors, desirably iPS cells, from a mixedpopulation of T-cells so as to generate an isolated, separated, orenriched population of T-cells, stimulating the isolated, separated orenriched population of T-cells so as to generate a stimulated populationof CD8+ expressing T-cells and/or CD4+ expressing T-cells, transducingthe stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells with a vector, wherein the vector encodes a chimericantigen receptor and a marker sequence, wherein said marker sequenceencodes a cell surface selectable marker, so as to generate a transducedpopulation of CD8+ expressing T-cells and/or CD4+ expressing T-cells,contacting the transduced population of CD8+ expressing T-cells and/orCD4+ expressing T-cells, for at least one day, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any timethat is within a range of times defined by any two of the aforementionedtime points, with at least one cytokine, which is preferably anexogenously added cytokine e.g., in addition to any cytokine eitherproduced by the T-cells or present in the media, so as to generate atransduced, cytokine-stimulated population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells, separating, enriching, or isolating thetransduced, cytokine-stimulated population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells by selection of the marker encoded by themarker sequence so as to generate an enriched, separated, or isolatedpopulation of transduced, cytokine-stimulated CD8+ expressing T-cellsand/or CD4+ expressing T-cells and propagating the enriched, separated,or isolated population of transduced, cytokine-stimulated CD8+expressing T-cells and/or CD4+ expressing T-cells for at least one day,such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 days or any time that is within a range of times defined byany two of the aforementioned time points, so as to obtain saidgenetically modified T-cells, which have a chimeric antigen receptor.

In some alternatives of the method, the vector further comprises a firstsequence encoding a leader sequence, a second sequence encoding a ligandbinding domain, a third sequence encoding a signaling domain and afourth sequence encoding a selectable marker sequence. In somealternatives of the method, the vector further comprises a sequenceencoding a spacer. In some alternatives of the method, the spacercomprises an IgG4 hinge. In some alternatives of the method, the vectoris a viral vector. In some alternatives of the method, the viral vectoris derived from simian virus 40, adenoviruses, adeno-associated virus(AAV), lentivirus, or retroviruses. In some alternatives of the method,the viral vector is a recombinant adenovirus, adeno-associated virus,lentivirus or retrovirus vector. In some alternatives of the method, theviral vector is a lentivirus vector. In some alternatives of the method,the marker sequence encodes a truncated epidermal growth factor receptor(EGFRt). In some alternatives of the method, the ligand binding domainof the chimeric antigen receptor comprises an antibody, or a bindingportion thereof. In some alternatives of the method, the ligand bindingdomain of the chimeric antigen receptor comprises a single chainvariable fragment (scFv), or a binding portion thereof. In somealternatives of the method, the ligand binding domain of the chimericantigen receptor comprises FMC63, or a binding portion thereof. In somealternatives of the method, the ligand binding domain of the chimericantigen receptor is specific for CD19. In some alternatives of themethod, the vector comprises a sequence encoding a maker sequence. Insome of these alternatives, the marker sequence is a truncated epidermalgrowth factor receptor (EGFRt).

“Codon optimization” as described herein, refers to the design processof altering codons to codons known to increase maximum proteinexpression efficiency. In some alternatives, codon optimization forexpression in human is described, wherein codon optimization can beperformed by using algorithms that are known to those skilled in the artso as to create synthetic genetic transcripts optimized for high mRNAand protein yield in humans. Programs containing algorithms for codonoptimization in humans are readily available. Such programs can include,for example, OptimumGene™ or GeneGPS® algorithms. Additionally humancodon optimized sequences can be obtained commercially, for example,from Integrated DNA Technologies.

In some alternatives, a method of making genetically modified T-cells,which have a chimeric antigen receptor is provided, wherein the methodcomprises separating, isolating, or enriching a CD8+ expressingpopulation of T-cells and/or a CD4+ expressing population of T-cells,such as T-cells that are derived from thymocytes or T-cells that arederived from engineered precursors, desirably iPS cells, from a mixedpopulation of T-cells so as to generate an isolated, separated, orenriched population of T-cells, stimulating the isolated, separated, orenriched population of T-cells so as to generate a stimulated populationof CD8+ expressing T-cells and/or CD4+ expressing T-cells, transducingthe stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells with a vector, wherein the vector encodes a chimericantigen receptor and a marker sequence, wherein said marker sequenceencodes a cell surface selectable marker, so as to generate a transducedpopulation of CD8+ expressing T-cells and/or CD4+ expressing T-cells,contacting the transduced population of CD8+ expressing T-cells and/orCD4+ expressing T-cells, for at least one day, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any timethat is within a range of times defined by any two of the aforementionedtime points, with at least one cytokine, which is preferably anexogenously added cytokine e.g., in addition to any cytokine eitherproduced by the T-cells or present in the media, so as to generate atransduced, cytokine-stimulated population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells, enriching, isolating, or separating thetransduced, cytokine-stimulated population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells by selection of the marker encoded by themarker sequence so as to generate an enriched, isolated, or separatedpopulation of transduced, cytokine-stimulated CD8+ expressing T-cellsand/or CD4+ expressing T-cells and propagating the enriched, separated,or isolated population of transduced, cytokine-stimulated CD8+expressing T-cells and/or CD4+ expressing T-cells for at least one day,such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 days or any time that is within a range of times defined byany two of the aforementioned time points, so as to obtain saidgenetically modified T-cells, which have a chimeric antigen receptor. Insome alternatives of the method, the vector further comprises a firstsequence encoding a leader sequence, a second sequence encoding a ligandbinding domain, a third sequence encoding a signaling domain and afourth sequence encoding a selectable marker sequence. In somealternatives of the method, the sequences of the vector are codonoptimized for expression in humans. In some alternatives, the sequencesof the vector are optimized to have selected codons specifically formaximal protein expression in human cells, which can increase theconcentration of proteins or CARs of a T-cell.

Optimization can also be performed to reduce the occurrence of secondarystructure in a polynucleotide. In some alternatives of the method,optimization of the sequences in the vector can also be performed toreduce the total GC/AT ratio. Strict codon optimization can lead tounwanted secondary structure or an undesirably high GC content thatleads to secondary structure. As such, the secondary structures affecttranscriptional efficiency. Programs such as GeneOptimizer can be usedafter codon usage optimization, for secondary structure avoidance and GCcontent optimization. These additional programs can be used for furtheroptimization and troubleshooting after an initial codon optimization tolimit secondary structures that can occur after the first round ofoptimization. Alternative programs for optimization are readilyavailable. In some alternatives of the method, the vector comprisessequences that are optimized for secondary structure avoidance and/orthe sequences are optimized to reduce the total GC/AT ratio and/or thesequences are optimized for expression in humans.

Marker domains can also provide important aspects to alternativesdescribed herein. Utilization of a marker domain on the cell surface canallow for transduced lymphocyte ablation in the event of a toxic eventassociated with administration or the presence of transduced T-cells.For example, in the case of an EGFRt marker sequence, full lengthantibodies to EGFR can be utilized to bind to cells expressing the EGFRand kill them via antibody dependent cell mediated cytotoxicity (ADCC).In some alternatives, an antibody specific for a marker domain can belinked to a cytotoxic agent, such as a radionuclide or a toxin, whichresults in a therapeutic capable of ablation or killing of thetransduced cells in vivo. In some alternatives, a marker sequence isprovided for enrichment and cell selection. In an exemplary alternativedescribed herein, an EGFRt marker sequence is used in a purification andenrichment method allowing for immunomagnetic selection against theEGFRt marker.

A “spacer” as described herein can refer to a polypeptide chain that canrange in length from a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239 or 240 amino acids or a length within arange defined by any two of the aforementioned lengths. A spacer cancomprise any 20 amino acids, for example, in any order to create adesirable length of polypeptide chain in a chimeric antigen receptor,which includes the amino acids arginine, histidine, lysine, asparticacid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine,glycine, proline, alanine, valine, isoleucine, methionine,phenylalanine, tyrosine and/or tryptophan. A spacer sequence can be alinker between the scFv and the transmembrane domain of the chimericantigen receptor. In some alternatives of the method of makinggenetically modified T-cells, which have a chimeric antigen receptor,the vector further comprises a sequence encoding a spacer. In somealternatives of the method, the spacer comprises a sequence with alength of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239 or 240 amino acids or a length within a range defined by anytwo of the aforementioned lengths. In some alternatives of the method,the spacer resides between the scFv and the transmembrane region of thechimeric antigen receptor.

“IgG4 hinge” as described herein, refers to the polypeptide domains thatare between the heavy chain and the light chains of the IgG4 antibody.In some alternatives, a method of making genetically modified T-cells,which have a chimeric antigen receptor is provided, wherein the methodcomprises separating, enriching, or isolating a CD8+ expressingpopulation of T-cells and/or a CD4+ expressing population of T-cells,such as T-cells that are derived from thymocytes or T-cells that arederived from engineered precursors, desirably iPS cells, from a mixedpopulation of T-cells so as to generate an isolated, separated, orenriched population of T-cells, stimulating the isolated, separated, orenriched population of T-cells so as to generate a stimulated populationof CD8+ expressing T-cells and/or CD4+ expressing T-cells, transducingthe stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells with a vector, wherein the vector encodes a chimericantigen receptor and a marker sequence, wherein said marker sequenceencodes a cell surface selectable marker, so as to generate a transducedpopulation of CD8+ expressing T-cells and/or CD4+ expressing T-cells,contacting the transduced population of CD8+ expressing T-cells and/orCD4+ expressing T-cells, for at least one day, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any timethat is within a range of times defined by any two of the aforementionedtime points, with at least one cytokine, which is preferably anexogenously added cytokine e.g., in addition to any cytokine eitherproduced by the T-cells or present in the media, so as to generate atransduced, cytokine-stimulated population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells, enriching the transduced,cytokine-stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells by selection of the marker encoded by the markersequence so as to generate an enriched, separated, or isolatedpopulation of transduced, cytokine-stimulated CD8+ expressing T-cellsand/or CD4+ expressing T-cells and propagating the enriched populationof transduced, cytokine-stimulated CD8+ expressing T-cells and/or CD4+expressing T-cells for at least one day, such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time thatis within a range of times defined by any two of the aforementioned timepoints, so as to obtain said genetically modified T-cells, which have achimeric antigen receptor.

In some alternatives of the method, the vector further comprises a firstsequence encoding a leader sequence, a second sequence encoding a ligandbinding domain, a third sequence encoding a signaling domain and afourth sequence encoding a selectable marker. In some alternatives ofthe method, the vector further comprises a sequence encoding a spacer.In some alternatives, the spacer comprises a hinge region of a humanantibody. In some alternatives of the method, the spacer comprises anIgG4 hinge. In some alternatives, the IgG4 hinge region is a modifiedIgG4 hinge. A “modified IgG4 hinge” as described herein can refer to ahinge region that can have at least 90%, 92%, 95%, or 100% sequenceidentity or a sequence identity within a range defined by any two of theaforementioned percentages, with a hinge region amino acid sequence asset forth in SEQ ID NO: 1 (SEQ ID NO: 1; ESKYGPPCPPCP), SEQ ID NO: 2(SEQ ID NO: 2; YGPPCPPCP), SEQ ID NO: 3 (SEQ ID NO: 3; KYGPPCPPCP), orSEQ ID NO: 4 (SEQ ID NO: 4; EVVKYGPPCPPCP). As mentioned above, any oneor more of these sequences can be codon optimized for expression inhumans and any one or more of these sequences can be optimized to reducesecondary structure or GC/AT ratio and any one or more of thesesequences can be consensus sequences generated from at least two isotypegenes.

A “transmembrane domain” is a region of a protein that is hydrophobicthat can reside in the bilayer of a cell to anchor a protein that isembedded to the biological membrane. Without being limiting, thetopology of the transmembrane domain can be a transmembrane alpha helix.In some alternatives of the method of making genetically modifiedT-cells, which have a chimeric antigen receptor, the vector comprises asequence encoding a transmembrane domain. In some alternatives of themethod, the transmembrane domain comprises a CD28 transmembrane sequenceor a fragment thereof that is a length of 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids or alength within a range defined by any two of the aforementioned lengths.In some alternatives of the method, the CD28 transmembrane sequence orfragment thereof comprise 28 amino acids in length. In some alternativesof the method, the transmembrane domain comprises the sequence set forthin SEQ ID NO: 5 (SEQ ID NO: 5; MFWVLVVVGGVLACYSLLVTVAFIIFWV).

A “signaling domain,” also known as a “Co-stimulatory domain” is anintracellular or cytoplasmic domain of a protein or a receptor proteinthat interacts with the interior of the cells and functions by relayinga signal. The portion of the protein that resides in the intracellularportion of the cell is also referred to as the “endodomain.” Thisinteraction can occur through the intracellular domain communicating viaspecific protein-protein or protein-ligand interactions with an effectormolecule or an effector protein, which in turn can send the signal alonga signal chain to its destination. The signaling or co-stimulatorydomain also refers to a signaling moiety that provides to T-cells asignal which, in addition to the primary signal provided by for instancethe CD3 zeta chain of the TCR/CD3 complex, mediates a T-cell response,such as, for example, an immune response, activation, proliferation,differentiation, cytokine secretion, cytolytic activity, perforin and/orgranzyme activity and the like. A signaling or co-stimulatory domain caninclude all or a portion of, but is not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, and/or B7-H3, and/or a ligand thatspecifically binds with CD83.

In some alternatives of the method of making genetically modifiedT-cells, which have a chimeric antigen receptor, the vector encoding thechimeric antigen receptor further comprises a sequence for aco-stimulatory domain, wherein the co-stimulatory domain is anintracellular signaling domain that interacts with other intracellularmediators to mediate a cell response including an immune response,activation, proliferation, differentiation, cytokine secretion,cytolytic activity, perforin and/or granzyme activity and the like. Insome alternatives of the method, the vector comprises a sequenceencoding a signaling domain. In some alternatives of the method, thesignaling domain comprises a 4-1BB domain and/or CD3-zeta domain. Insome alternatives, the 4-1BB domain comprises the sequence set forth inSEQ ID NO: 6 (SEQ ID NO: 6; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).In some alternatives, the CD3-zeta domain comprises the sequence setforth in SEQ ID NO: 7 (SEQ ID NO: 7;RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR).

A “chimeric antigen receptor” (CAR) described herein, also known aschimeric T-cell receptor, refers to an artificial T-cell receptor or agenetically engineered receptor, which grafts a desired specificity ontoan immune effector cell. These receptors can be used to graft thespecificity of a monoclonal antibody or a binding portion thereof onto aT-cell, for example; with transfer of the coding sequence for the CAR toa recipient cell being facilitated by a retroviral vector. The structureof the CAR can comprise single-chain variable fragments (scFv) derivedfrom monoclonal antibodies, fused to CD3-zeta transmembrane andendodomain. Such molecules result in the transmission of a zeta signalin response to recognition by the scFv of its target.

In some alternatives, a method of making genetically modified T-cells,which have a chimeric antigen receptor is provided, wherein the methodcomprises separating, isolating, or enriching a CD8+ expressingpopulation of T-cells and/or a CD4+ expressing population of T-cells,such as T-cells that are derived from thymocytes or T-cells that arederived from engineered precursors, desirably iPS cells, from a mixedpopulation of T-cells, from a mixed population of T-cells, so as togenerate an isolated, separated, or enriched population of T-cells,stimulating the isolated, separated, or enriched population of T-cellsso as to generate a stimulated population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells, transducing the stimulated population ofCD8+ expressing T-cells and/or CD4+ expressing T-cells with a vector,wherein the vector encodes a chimeric antigen receptor and a markersequence, wherein said marker sequence encodes a cell surface selectablemarker, so as to generate a transduced population of CD8+ expressingT-cells and/or CD4+ expressing T-cells, contacting the transducedpopulation of CD8+ expressing T-cells and/or CD4+ expressing T-cellswith at least one cytokine, such as for at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time thatis within a range of times defined by any two of the aforementioned timepoints, so as to generate a transduced, cytokine-stimulated populationof CD8+ expressing T-cells and/or CD4+ expressing T-cells, enriching,separating or isolating the transduced, cytokine-stimulated populationof CD8+ expressing T-cells and/or CD4+ expressing T-cells by selectionof the marker encoded by the marker sequence so as to generate anenriched, separated, or isolated population of transduced,cytokine-stimulated CD8+ expressing T-cells and/or CD4+ expressingT-cells and propagating the enriched, separated, or isolated populationof transduced, cytokine-stimulated CD8+ expressing T-cells and/or CD4+expressing T-cells for at least one day, such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time thatis within a range of times defined by any two of the aforementioned timepoints, so as to obtain said genetically modified T-cells, which have achimeric antigen receptor. In some alternatives of the method, thechimeric antigen receptor is specific for CD19.

These artificial T-cell receptors, or CARs, can be used as a therapy forcancer or a viral infection using a technique called “adoptive celltransfer.” T-cells are removed from a patient and modified so that theyexpress receptors specific for a molecule displayed on a cancer cell ora virus, or a virus-infected cell. The genetically engineered T-cells,which can then recognize and kill the cancer cells or the virus infectedcells or promote clearance of the virus, are reintroduced into thepatient. In some alternatives, a method of treating, inhibiting, orameliorating a disease in a subject in need thereof is provided.

In some aspects, the method can comprise administering to the at leastone composition or product combination, wherein the at least onecomposition or product combination comprises a population of geneticallymodified T-cells, wherein the population of genetically modified T-cellscomprises a plurality of affinity selected CD8+ and/or CD4+ expressingT-cells, in the absence of, enriched from, substantially separated fromor substantially isolated from CD8− and/or CD4− expressing T-cells,wherein said plurality of affinity selected CD8+ and/or CD4+ expressingT-cells have stimulated CD2, CD3, CD4 and/or CD28 receptors, whereinsaid plurality of affinity selected CD8+ and/or CD4+ expressing T-cellsfurther comprise a gene encoding a chimeric antigen receptor and a cellsurface selectable marker and, wherein said plurality of affinityselected CD8+ and/or CD4+ expressing T-cells have been re-stimulatedwith at least one cytokine, such as for at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any time thatis within a range of times defined by any two of the aforementioned timepoints. In some alternatives, the at least one composition or productcombination is administered to said subject by adoptive cell transfer.

An “antibody” as described herein refers to a large Y-shape proteinproduced by plasma cells that is used by the immune system to identifyand neutralize foreign objects such as bacteria and viruses. Theantibody protein can comprise four polypeptide chains; two identicalheavy chains and two identical light chains connected by disulfidebonds. Each chain is composed of structural domains calledimmunoglobulin domains. These domains can contain 70-110 amino acids andare classified into different categories according to their size andfunction. In some alternatives, the ligand binding domain is an antibodyfragment, desirably, a binding portion thereof. In some alternatives,the antibody fragment or binding portion thereof present on a CAR isspecific for CD19.

A “single chain variable fragment” or scFv is a fusion protein thatcomprises the variable regions of the heavy chain (VH) and the lightchains (VL) of an immunoglobulin that is connected with a short linkerpeptide. Without being limiting, the linker can comprise glycine forflexibility and hydrophilic amino acids, for example serine or threoninefor solubility. The linker can connect the N-terminus of the VH with theC-terminus of the VL or it can connect the C-terminus of the VH with theN-terminus of the VL. In some alternatives, the ligand binding domainpresent on a CAR is a single chain variable fragment (scFv). In somealternatives, the scFv domain present on a CAR is specific for a CD19present on a tumor cell.

“FMC63” is a CD19 specific monoclonal antibody. CD19 is a protein thatis found on the surface of white blood cells and can assemble with theantigen receptor of B lymphocytes in order to decrease the threshold forantigen receptor-dependent stimulation. CD19 is expressed on folliculardendritic cells and B cells. CD19 is present on B cells from earliestrecognizable B-lineage cells during development to B-cell blasts but islost on maturation to plasma cells. CD19 primarily acts as a B cellco-receptor in conjunction with CD21 and CD81. Upon activation, thecytoplasmic tail of CD19 becomes phosphorylated, which leads to bindingby Src-family kinases and recruitment of PI-3 kinase. As on T-cells,several surface molecules form the antigen receptor and form a complexon B lymphocytes.

Mutations in CD19 are associated with severe immunodeficiency syndromescharacterized by diminished antibody production. For example, aberrantexpression of CD19 is a marker of monocytic lineage in acute myelogenousleukemia. Since CD19 is a hallmark of B-cells, the protein can be usedto diagnose cancers that arise from this type of cell, notably B-celllymphomas. Since 2011, treatments targeting CD19 have begun to entertrials. Most current experimental anti-CD19 drugs in development work byexploiting the presence of CD19 to direct treatment specifically towardsB-cell cancers. However, it is now emerging that the protein plays anactive role in driving the growth of these cancers, by stabilizing theconcentrations of the MYC oncoprotein. Thus, CD19 and its downstreamsignaling can be attractive therapeutic targets.

In some alternatives, a method of making genetically modified T-cells,which have a chimeric antigen receptor is provided, wherein the methodcomprises separating, enriching, isolating, or purifying a population ofCD8+ expressing T-cells and/or a population of CD4+ expressing T-cells,such as T-cells that are derived from thymocytes or T-cells that arederived from engineered precursors, desirably iPS cells, from a mixedpopulation of T-cells, from a mixed population of T-cells, from a mixedpopulation of T-cells so as to generate an isolated, separated,enriched, or purified population of T-cells, stimulating the isolated,separated, enriched, or purified population of T-cells so as to generatea stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells, transducing the stimulated population of CD8+expressing T-cells and/or CD4+ expressing T-cells with a vector, whereinthe vector encodes a chimeric antigen receptor and a marker sequence,wherein said marker sequence encodes a cell surface selectable marker,so as to generate a transduced population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells, contacting the transduced population ofCD8+ expressing T-cells and/or CD4+ expressing T-cells with at least onecytokine, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 days or any time that is within arange of times defined by any two of the aforementioned time points, soas to generate a transduced, cytokine-stimulated population of CD8+expressing T-cells and/or CD4+ expressing T-cells, enriching thetransduced, cytokine-stimulated population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells by selection of the marker so as togenerate an enriched population of transduced, cytokine-stimulated CD8+expressing T-cells and/or CD4+ expressing T-cells and propagating theenriched population of transduced, cytokine-stimulated CD8+ expressingT-cells and/or CD4+ expressing T-cells for at least one day, such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20days or any time that is within a range of times defined by any two ofthe aforementioned time points, so as to obtain said geneticallymodified T-cells, which have a chimeric antigen receptor.

In some alternatives of the method, the vector further comprises a firstsequence encoding a leader sequence, a second sequence encoding a ligandbinding domain, a third sequence encoding a signaling domain and afourth sequence encoding a selectable marker sequence. In somealternatives of the method, the vector further comprises a sequenceencoding a spacer. In some alternatives of the method, the ligandbinding domain of the chimeric antigen receptor comprises an antibody,or a binding portion thereof. In some alternatives of the method, theligand binding domain of the chimeric antigen receptor comprises asingle chain variable fragment (scFv), or a binding portion thereof. Insome alternatives of the method, the ligand binding domain of thechimeric antigen receptor comprises FMC63, or a binding portion thereof.In some alternatives of the method, the ligand binding domain of thechimeric antigen receptor is specific for CD19.

“Purification” of T-cells as described herein, refers to the isolationof highly purified, functional T-cells for research or for methods forgenerating T-cells for therapy. “Isolation” of T-cells, or “separating”of T-cells, as described herein, refers to a procedure of isolating orseparating the desired T-cells, T-cell populations, or subpopulationsfrom contaminating cell populations or other components, for example byusing techniques, such as indirect panning. In this method, cells can beisolated, separated, or selected by their capacity to bind to anantibody that is attached to a support, such as a plastic or polycarbonate surface, bead, particle, plate, or well. Cells can bind on thebasis of particular cell surface markers. In some cases the desiredT-cell populations are “enriched” meaning that the population of thedesired T-cells is greater after enrichment than that of the nativepopulation from which the T-cells originated.

In some alternatives of the method of making a genetically modifiedT-cell, the CD8+ expressing population of T-cells and/or CD4+ expressingpopulation of T-cells are isolated, enriched, purified or separated fromundesired components using CD8 specific antibodies and/or CD4 specificantibodies. In some alternatives of the method, the CD8+ expressingpopulation of T-cells and/or CD4+ expressing population of T-cells areisolated, enriched, or purified using flow cytometry. In somealternatives, the CD8+ expressing population of T-cells and/or CD4+expressing population of T-cells are isolated, enriched, purified, orseparated using immunomagnetic selection. In some alternatives of themethod, the CD8+ expressing population of T-cells and/or CD4+ expressingpopulation of T-cells are isolated, enriched, purified, or separatedusing antibodies against CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57,CD137, CD27, CD28, CD8, CD4, and/or CD62L. In some alternatives of themethod, the CD8+ expressing population of T-cells and/or CD4+ expressingpopulation of T-cells are isolated, enriched, purified, or separated byusing antibodies against CD27, CD28 and/or CD62L. In some alternatives,the method is performed with isolated, enriched, or separated CD4+expressing T-cells in the absence of or in greater abundance than theamount of CD8+ expressing cells in the mixed population of T-cells usedfor the isolation, enrichment, or purification. In some alternatives,the method is performed with isolated, enriched, or separated CD8+ inthe absence of or in greater abundance than the amount of CD4+expressing cells in the mixed population of T-cells used for theisolation, enrichment, or purification.

“Stimulation” or activation of T-cells refers to the method of inducinga T-cell to initiate a response, such as a signal transduction response,e.g., proliferation, while preserving T-cell viability and immunefunction. For example, simultaneous signaling to TCR/CD3 and CD8 cantrigger a physiological activation and expansion of human T-cells.Following appropriate stimulation, T-cells may proliferate extensivelyin vitro. In some alternatives, a method of making genetically modifiedT-cells, which have a chimeric antigen receptor is provided wherein themethod comprises isolating, separating, enriching, or purifying a CD8+expressing population of T-cells and/or a CD4+ expressing population ofT-cells, such as T-cells that are derived from thymocytes or T-cellsthat are derived from engineered precursors, desirably iPS cells, from amixed population of T-cells, so as to generate an isolated, separated,purified, or enriched population of T-cells, stimulating the isolated,separated, purified, or enriched population of T-cells so as to generatea stimulated population of CD8+ expressing T-cells and/or CD4+expressing T-cells, transducing the stimulated population of CD8+expressing T-cells and/or CD4+ expressing T-cells with a vector, whereinthe vector encodes a chimeric antigen receptor and a marker sequence,wherein said marker sequence encodes a cell surface selectable marker,so as to generate a transduced population of CD8+ expressing T-cellsand/or CD4+ expressing T-cells, contacting the transduced population ofCD8+ expressing T-cells and/or CD4+ expressing T-cells with at least onecytokine, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 days or any time that is within arange of times defined by any two of the aforementioned time points, soas to generate a transduced, cytokine-stimulated population of CD8+expressing T-cells and/or CD4+ expressing T-cells, enriching,separating, or isolating the transduced, cytokine-stimulated populationof CD8+ expressing T-cells and/or CD4+ expressing T-cells by selectionof the marker encoded by the marker sequence so as to generate anenriched population of transduced, cytokine-stimulated CD8+ T-cellsand/or CD4+ T-cells and propagating the enriched, isolated, or separatedpopulation of transduced, cytokine-stimulated CD8+ expressing T-cellsand/or CD4+ expressing T-cells for at least one day, such as for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 days or any time that is within a range of times defined by anytwo of the aforementioned time points, so as to obtain said geneticallymodified T-cells, which have a chimeric antigen receptor. In somealternatives, the stimulating is performed with an antibody-boundsupport, such as a bead or particle. In some alternatives, thestimulating is performed with an antibody-bound support comprisinganti-TCR, anti-CD2, anti-CD3, anti-CD4 and/or anti-CD28 antibodies. Insome alternatives, the stimulating is performed with an antibody-boundsupport comprising anti-CD3 and/or anti-CD28 antibodies. In somealternatives, the method further comprises removing the antibody-boundsupport, such as beads or particles.

“T-cells” or “T lymphocytes” as used herein can be from any mammal,preferably a primate, including monkeys or humans, a companion animalsuch as a dog, cat, or horse, or a domestic animal, such as a sheep,goat, or cattle. In some alternatives the T-cells are allogeneic (fromthe same species but different donor) as the recipient subject; in somealternatives the T-cells are autologous (the donor and the recipient arethe same); in some alternatives the T-cells arc syngeneic (the donor andthe recipients are different but are identical twins).

“CD8+ expressing T-cell” or “CD8+ T-cell,” used synonymously throughout,is also known as a TC, cytotoxic T lymphocyte, CTL, T-killer cell,cytolytic T-cell or killer T-cell. As described herein, CD8+ T-cells areT-lymphocytes that can kill cancer cells, virally infected cells, ordamaged cells. CD8+ T-cells express T-cell receptors (TCRs) that canrecognize a specific antigen. CD8+ T-cells express CD8 on the surface.CD8+ expressing T-cells have the ability to make some cytokines, howeverthe amounts of cytokines made by CD8+ T-cells are not at a concentrationthat promotes, improves, contributes to, or induces engraftment fitness.

“CD4+ expressing T-cell,” or “CD4+ T-cell,” used synonymouslythroughout, is also known as T helper cells, which play an importantrole in the immune system, and in the adaptive immune system. CD4+T-cells also help the activity of other immune cells by releasing T-cellcytokines. These cells help, suppress or regulate immune responses. Theyare essential in B cell antibody class switching, in the activation andgrowth of cytotoxic T-cells, and in maximizing bactericidal activity ofphagocytes, such as macrophages. CD4+ expressing T-cells have theability to make some cytokines, however the amounts of cytokines made byCD4+ T-cells are not at a concentration that promotes, improves,contributes to, or induces engraftment fitness.

Mature T cells express the surface protein CD4 and are referred to asCD4+ T-cells. CD4+ T-cells are generally treated as having a pre-definedrole as helper T-cells within the immune system. For example, when anantigen-presenting cell expresses an antigen on MHC class II, a CD4+cell will aid those cells through a combination of cell to cellinteractions (e.g. CD40 and CD40L) and through cytokines Nevertheless,there are rare exceptions; for example, sub-groups of regulatoryT-cells, natural killer cells, and cytotoxic T-cells express CD4. All ofthe latter CD4+ expressing T-cell groups are not considered T helpercells.

“Central memory” T-cell (or “T_(CM)”) as used herein refers to anantigen experienced CTL that expresses CD62L or CCR-7 and CD45RO on thesurface thereof, and does not express or has decreased expression ofCD45RA as compared to naïve cells. In some alternatives, central memorycells are positive for expression of CD62L, CCR7, CD28, CD127, CD45RO,and/or CD95, and have decreased expression of CD54RA as compared tonaïve cells.

“Effector memory” T-cell (or “T_(EM)”) as used herein refers to anantigen experienced T-cell that does not express or has decreasedexpression of CD62L on the surface thereof as compared to central memorycells, and does not express or has decreased expression of CD45RA ascompared to naïve cell. In some alternatives, effector memory cells arenegative for expression of CD62L and/or CCR7, as compared to naïve cellsor central memory cells, and have variable expression of CD28 and/orCD45RA.

“Naïve” T-cells as used herein refers to a non-antigen experienced Tlymphocyte that expresses CD62L and/or CD45RA, and/or does not expressCD45RO− as compared to central or effector memory cells. In somealternatives, naïve CD8+ T lymphocytes are characterized by theexpression of phenotypic markers of naïve T-cells including CD62L, CCR7,CD28, CD127, and/or CD45RA.

“Effector” “T_(E)” T-cells as used herein refers to a antigenexperienced cytotoxic T lymphocyte cells that do not express or havedecreased expression of CD62L, CCR7, CD28, and are positive for granzymeB and/or perforin, as compared to central memory or naïve T-cells.

“Engraftment fitness” as described herein, refers to the ability of acell to grow and proliferate after the cells have entered the body,e.g., blood stream, through adoptive transfer. Engraftment can usuallyoccur within two to four weeks after the transfer. Engraftment can bemonitored by checking blood counts for a specific cell on a frequentbasis. In some alternatives of the method of treating, inhibiting, orameliorating a disease in a subject is provided, the method can compriseadministering a composition or product combination comprising thegenetically modified T-cells, as described herein. In some alternatives,the method can further comprise monitoring the subject by checking theblood counts for the genetically modified T-cells that expresses achimeric antigen receptor e.g., by identifying the presence or absenceof a marker associated with the transferred T-cells.

T-cells with improved engraftment fitness may have specific markers onthe cell surface that confer generation and long term maintenance ofT-cell immunity. There are several proteins that are known for T-cellactivation and survival. CD28 is a protein expressed on T-cells thatprovide co-stimulatory signals required for T-cell activation andsurvival. CD27 is required for generation and long-term maintenance ofT-cell immunity. It binds to ligand CD70, and plays a key role inregulating B-cell activation and immunoglobulin synthesis. L-selectin,also known as CD62L is a cell adhesion molecule found on lymphocytes.L-selectin functions as a “homing receptor” for lymphocytes or T-cellsto enter secondary lymphoid tissues via high endothelial venues. Ligandspresent on endothelial cells will bind to lymphocytes expressingL-selectin, slowing lymphocyte trafficking through the blood, and canfacilitate entry into a secondary lymphoid organ at that point. In somealternatives, of the method of making genetically modified T-cells,which have a chimeric antigen receptor, the T-cells comprise at leastone receptor that promotes, induces, improves, or contributes toengraftment fitness. In some alternatives, the at least one receptor isCD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/orCD62L. In some alternatives, the at least one receptor is CD27, CD28and/or CD62L. In some alternatives provided herein, methods aredescribed in which CD4+ and CD8+ expressing T-cells are geneticallymodified by transduction of genetic material in the form of a vectorsuch that the genetically modified CD4+ and CD8+ expressing T-cellsexpresses a specific chimeric antigen receptor. In some alternatives ofthe genetically modified CD4+ and CD8+ T-cells, the genetically modifiedCD4+ and CD8+ T-cells are further modified to improve or enhanceengraftment fitness.

“Cytokines” as described herein, refers to small proteins (5-25 kDa)that are important in cell signaling. Cytokines are released by cellsand affect the behavior of other cells, and sometimes the releasing cellitself, such as a T-cell. Cytokines can include, for example,chemokines, interferons, interleukins, lymphokines, and tumor necrosisfactor. Cytokines can be produced by a broad range of cells, which caninclude, for example, immune cells like macrophages, B lymphocytes, Tlymphocytes and mast cells, as well as, endothelial cells, fibroblasts,and various stromal cells.

Cytokines can act through receptors, and are important in the immunesystem as the cytokines can modulate the balance between humoral andcell-based immune responses, and they can regulate the maturation,growth, and responsiveness of particular cell populations. Somecytokines enhance or inhibit the action of other cytokines in complexways. Without being limiting, cytokines can include, for example,Acylation stimulating protein, Adipokine, Albinterferon, CCL1, CCL11,CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20,CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL5,CCL6, CCL7, CCL8, CCL9, Chemokine, Colony-stimulating factor, CX3CL1,CX3CR1, CXCL1, CXCL10, CXCL11, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17,CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL9, Erythropoietin, Gc-MAF,Granulocyte colony-stimulating factor, Granulocyte macrophagecolony-stimulating factor, Hepatocyte growth factor, IL 10 family ofcytokines, IL 17 family of cytokines, IL1A, IL1B, Inflammasome,Interferome, Interferon, Interferon beta 1a, Interferon beta 1b,Interferon gamma, Interferon type I, Interferon type II, Interferon typeIII, Interleukin, Interleukin 1 family, Interleukin 1 receptorantagonist, Interleukin 10, Interleukin 12, Interleukin 12 subunit beta,Interleukin 13, Interleukin 15, Interleukin 16, Interleukin 2,Interleukin 23, Interleukin 23 subunit alpha, Interleukin 34,Interleukin 35, Interleukin 6, Interleukin 7, Interleukin 8, Interleukin36, Leukemia inhibitory factor, Leukocyte-promoting factor, Lymphokine,Lymphotoxin, Lymphotoxin alpha, Lymphotoxin beta, Macrophagecolony-stimulating factor, Macrophage inflammatory protein,Macrophage-activating factor, Monokine, Myokine, Myonectin, Nicotinamidephosphoribosyltransferase, Oncostatin M, Oprelvekin, Platelet factor 4,Proinflammatory cytokine, Promegapoietin, RANKL, Stromal cell-derivedfactor 1, Talimogene laherparepvec, Tumor necrosis factor alpha, Tumornecrosis factors, XCL1, XCL2, GM-CSF, and/or XCR1. In some alternativesof the method of making genetically modified T-cells, a transducedpopulation of CD8+ expressing T-cells and/or CD4+ expressing T-cells iscontacted with at least one cytokine so as to generate a transduced,cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells. Insome alternatives of the method, the at least one cytokine utilizedcomprises GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2, and/or IL-21. In somealternatives, the period of contact with the cytokine is at least oneday, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 days or any time that is within a range oftimes defined by any two of the aforementioned time points.

“Interleukins” or IL as described herein, are cytokines that the immunesystem depends largely upon. Examples of interleukins, which can beutilized herein, for example, include IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, 11-7, IL-8/CXCL8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25,IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35,and/or IL-36. Contacting T-cells with interleukins can have effects thatpromote, support, induce, or improve engraftment fitness of the cells.IL-1, for example can function in the maturation & proliferation ofT-cells. IL-2, for example, can stimulate growth and differentiation ofT-cell response. IL-3, for example, can promote differentiation andproliferation of myeloid progenitor cells. IL-4, for example, canpromote proliferation and differentiation. IL-7, for example, canpromote differentiation and proliferation of lymphoid progenitor cells,involved in B, T, and NK cell survival, development, and homeostasis.IL-15, for example, can induce production of natural killer cells.IL-21, for example, co-stimulates activation and proliferation of CD8+T-cells, augments NK cytotoxicity, augments CD40-driven B cellproliferation, differentiation and isotype switching, and promotesdifferentiation of Th17 cells.

In some alternatives, a method of making a genetically modified T-cellis provided, wherein the method comprises purifying, separating,enriching, or isolating a CD8+ population of T-cells and/or a CD4+population of T-cells from a mixed population of T-cells, such asT-cells that are derived from thymocytes or T-cells that are derivedfrom engineered precursors, desirably iPS cells, from a mixed populationof T-cells, so as to generate an isolated, separated, enriched, orpurified population of T-cells, stimulating the isolated, separated,enriched, or purified population of T-cells so as to generate astimulated population of CD8+ T-cells and/or CD4+ T-cells, transducingthe stimulated population of CD8+ T-cells and/or CD4+ T-cells with avector, wherein the vector encodes a chimeric antigen receptor and amarker sequence, wherein said marker sequence encodes a cell surfaceselectable marker, so as to generate a transduced population of CD8+T-cells and/or CD4+ T-cells, contacting the transduced population ofCD8+ T-cells and/or CD4+ T-cells with at least one cytokine so as togenerate a transduced, cytokine-stimulated population of CD8+ T-cellsand/or CD4+ T-cells, enriching, isolating, or separating the transduced,cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells byselection of the marker encoded by the marker sequence so as to generatean enriched, isolated or separated population of transduced,cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells and propagating theenriched, isolated, or separated population of transduced,cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells for at least oneday, such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 days or any time that is between a range oftimes defined by any two of the aforementioned time points, so as toobtain said genetically modified T-cells, which have a chimeric antigenreceptor. In some alternatives, the at least one cytokine comprisesGM-CSF, IL-7, IL-12, IL-18, IL-15, IL-2, and/or IL-21. In somealternatives, the at least one cytokine comprises IL/7, IL-15 and/orIL-21. In some alternatives, the at least one cytokine comprises IL-2,IL-15 and/or IL-21. In some alternatives of the method, the cytokinecontacting is performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 days or a period of time within a rangedefined by any two of these time periods. In some alternatives, theaddition of the at least one cytokine improves, enhances, promotes, orinduces engraftment fitness. In some alternatives, the enriched CD4+expressing T-cells are contacted, for example propagated, in 5 ng/mLrecombinant human IL-7 (rhIL-7) and/or 0.5 ng/mL recombinant human IL-15(rhIL-15). In some alternatives, the enriched CD8+ expressing T-cellsare contacted, for example propagated, in 50 U/mL recombinant human IL-2(rhIL-2) and/or 0.5 ng/mL recombinant human IL-15 (rhIL-15). In morealternatives, the enriched CD4+ expressing T-cells are contacted, forexample propagated, in 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5ng/mL, 0.6 ng/mL, 0.7 ng/mL, 0.8 ng/mL, 0.9 ng/mL, or 1.0 ng/mL rhIL-7or in an amount that is within a range defined by any two of theaforementioned amounts of rhIL-7 and/or 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL,0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL, 0.8 ng/mL, 0.9 ng/mL, or 1.0ng/mL rhIL-15 or in an amount that is within a range defined by any twoof the aforementioned amounts of rhIL-15. In more alternatives, theenriched CD8+ expressing T-cells are contacted, for example propagated,in 10 U/mL, 20 U/mL, 30 U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80U/mL, 90 U/mL, or 100 U/mL of rhIL-2 or in an amount that is within arange defined by any two of the aforementioned amounts of rhIL-2 and/or0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7ng/mL, 0.8 ng/mL, 0.9 ng/mL, or 1.0 ng/mL rhIL-7 and/or 0.1 ng/mL, 0.2ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.6 ng/mL, 0.7 ng/mL, 0.8 ng/mL,0.9 ng/mL, or 1.0 ng/mL rhIL-15 or in an amount that is within a rangedefined by any two of the aforementioned amounts of rhIL-15. In somealternatives the aforementioned cytokines and amounts areco-administered to the T-cells, e.g., in a mixture or added shortlyafter one another, and in other alternatives, the aforementionedcytokines are contacted with the T cells separately, e.g., separated bya time period of 1-10 minutes or 1-10 hours.

“Enriched” and “depleted” are also used herein to describe amounts ofcell types in a mixture refers to the subjecting of the mixture of thecells to a process or step, which results in an increase in the numberof the “enriched” type and a decrease in the number of the “depleted”cells. Thus, depending upon the source of the original population ofcells subjected to the enriching process, a mixture or composition cancontain 60, 70, 80, 90, 95, or 99 percent or more, or any value within arange defined by any two of these values (in number or count) of the“enriched” cells and 40, 30, 20, 10, 5 or 1 percent or less or any valuewithin a range defined by any two of these values (in number or count)of the “depleted” cells. In some alternatives of the method of makinggenetically modified T-cells, enriching the transduced,cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells byaffinity selection of a cell surface marker is contemplated, such thatan enriched population of transduced, cytokine-stimulated CD8+ T-cellsand/or CD4+ T-cells is generated.

“Propagating cells” or propagation refers to steps to allowproliferation, expansion, growth and reproduction of cells. For example,cultures of CD8+ T-cells and CD4+ T-cells can typically be incubatedunder conditions that are suitable for the growth and proliferation of Tlymphocytes. In some alternatives of the method of making geneticallymodified T-cells, which have a chimeric antigen receptor, the CD4+expressing T-cells are propagated for at least 1 day and may bepropagated for 20 days, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 days or for a period that is within arange defined by any two of the aforementioned time periods. In somealternatives of the method of making genetically modified T-cells, whichhave a chimeric antigen receptor, the CD8+ expressing T-cells arepropagated for at least 1 day and may be propagated for 20 days, such as1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20days or for a period that is within a range defined by any two of theaforementioned time periods.

“Affinity selection,” as described herein, refers to the selection of aspecific molecule or cell having a selectable cell surface marker bybinding to the molecule or marker or an epitope present thereon with abinding affinity agent, which allows for one to select out the specificmolecule or cell of interest. Affinity selection can be performed by,for example, antibodies, conjugated antibodies, lectins, aptamers,and/or peptides. In some alternatives, of the method of makinggenetically modified T-cells, the separating of the CD8+ population ofT-cells and/or a CD4+ population of T-cells from a mixed population ofT-cells is performed by affinity selection for T-cells having an epitopepresent on CD8 and/or CD4. In some alternatives of the method, anti-CD8or anti-CD4 antibodies or binding portions thereof are used to selectout the cells of interest. In some alternatives of the method, theseparating of the CD8+ population of T-cells and/or a CD4+ population ofT-cells from a mixed population of T-cells is performed by flowcytometry. In some alternatives of the method, the separating of theCD8+ population of T-cells and/or a CD4+ population of T-cells from amixed population of T-cells is performed by immuno-magnetic selection.In some alternatives of the method, the anti-CD8 or the anti-CD4antibodies are conjugated to a solid support such as, for example, aninert bead or an inert particle.

“T cell precursors” as described herein refers to lymphoid precursorcells that can migrate to the thymus and become T cell precursors, whichdo not express a T cell receptor. All T cells originate fromhematopoietic stem cells in the bone marrow. Hematopoietic progenitors(lymphoid progenitor cells) from hematopoietic stem cells populate thethymus and expand by cell division to generate a large population ofimmature thymocytes. The earliest thymocytes express neither CD4 norCD8, and are therefore classed as double-negative (CD4 CD8) cells. Asthey progress through their development, they become double-positivethymocytes (CD4⁺CD8⁺), and finally mature to single positive (CD4⁺CD8⁻or CD4⁺CD8⁺) thymocytes that are then released from the thymus toperipheral tissues. About 98% of thymocytes die during the developmentprocesses in the thymus by failing either positive selection or negativeselection, whereas the other 2% survive and leave the thymus to becomemature immunocompetent T cells.

The double negative (DN) stage of the precursor T cell is focused onproducing a functional β-chain whereas the double positive (DP) stage isfocused on producing a functional α-chain, ultimately producing afunctional αβ T cell receptor. As the developing thymocyte progressesthrough the four DN stages (DN1, DN2, DN3, and DN4), the T cellexpresses an invariant α-chain but rearranges the β-chain locus. If therearranged β-chain successfully pairs with the invariant α-chain,signals are produced which cease rearrangement of the β-chain (andsilence the alternate allele) and result in proliferation of the cell.Although these signals require this pre-TCR at the cell surface, theyare dependent on ligand binding to the pre-TCR. These thymocytes willthen express both CD4 and CD8 and progresses to the double positive (DP)stage where selection of the α-chain takes place. If a rearrangedβ-chain does not lead to any signaling (e.g. as a result of an inabilityto pair with the invariant α-chain), the cell may die by neglect (lackof signaling).

“Hematopoietic stem cells” or “HSC” as described herein, are precursorcells that can give rise to myeloid cells such as, for example,macrophages, monocytes, macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells andlymphoid lineages (such as, for example, T-cells, B-cells, NK-cells).HSCs have a heterogeneous population in which three classes of stemcells exist, which are distinguished by their ratio of lymphoid tomyeloid progeny in the blood (L/M).

“Cryopreservation” is a process of preserving cells, whole tissues, orsubstances susceptible to damage by cooling to sub-zero temperatures. Atthe low temperatures any enzymatic or chemical activity, which can causedamage to the cells, tissue or substances in question are effectivelystopped. Cryopreservation methods seek to reach low temperatures withoutcausing additional damage caused by the formation of ice duringfreezing. Traditional cryopreservation has relied on coating thematerial to be frozen with a class of molecules termed cryoprotectants.New methods are constantly being investigated due to the inherenttoxicity of many cryoprotectants. Cryopreservation methods are known tothose skilled in the art. In some alternatives of the method of makinggenetically modified T-cells, which have a chimeric antigen receptor,the method can further comprise cryopreserving the genetically modifiedT-cells.

In some alternatives, a population of genetically modified T-cells isprovided, wherein the population of genetically modified T-cells, suchas T-cells that are derived from thymocytes or T-cells that are derivedfrom engineered precursors, desirably iPS cells, comprises a pluralityof affinity selected CD8+ and/or CD4+ expressing T-cells, in the absenceof, enriched over, or isolated from CD8− and/or CD4− T-cells, whereinsaid plurality of affinity selected CD8+ and/or CD4+ T-cells havestimulated CD2, CD3, CD4 and/or CD28 receptors, wherein said pluralityof affinity selected CD8+ and/or CD4+ T-cells further comprise a geneencoding a chimeric antigen receptor and a cell surface selectablemarker and, wherein said plurality of affinity selected CD8+ and/or CD4+expressing T-cells have been re-stimulated with at least one cytokine,such as for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 days or any time that is within a range of timesdefined by any two of the aforementioned time points. In somealternatives, the selected CD8+ and/or CD4+ T-cells are generated bymethods provided herein. In some alternatives, the at least one cytokinecomprises GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2, and/or IL-21. In somealternatives, the at least one cytokine comprises IL/7, IL-15 and/orIL-21. In some alternatives, the at least one cytokine comprises IL-2,IL-15 and/or IL-21. In some alternatives of the population ofgenetically modified T-cells, the plurality of affinity selected CD8+and/or CD4+ T-cells further comprise at least one receptor thatpromotes, induces, enhances or contribute to engraftment fitness. Insome alternatives, the at least one receptor that promotes, enhances,induces, or contributes to engraftment fitness is CD45 RA, CD45 RO,CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In somealternatives, the at least one receptor that promotes, enhances,contributes to, or induces engraftment fitness is CD27, CD28 and/orCD62L. In some alternatives, the plurality of affinity selected CD8+and/or CD4+ expressing T-cells further comprises a vector having a firstsequence encoding a leader sequence, a second sequence encoding a ligandbinding domain, a third sequence encoding a signaling domain and afourth sequence encoding a selectable marker. In some alternatives, thevector further comprises a sequence encoding a spacer. In somealternatives, the spacer comprises an IgG4 hinge. In some alternatives,the vector is a viral vector. In some alternatives, the viral vector isderived from simian virus 40, adenoviruses, adeno-associated virus(AAV), lentivirus, or retroviruses. In some alternatives, the viralvector is a recombinant adenovirus, adeno-associated virus, lentivirusor retrovirus vector. In some alternatives, the viral vector is alentivirus vector. In some alternatives, the cell surface selectablemarker codes for a truncated epidermal growth factor receptor (EGFRt).In some alternatives, the ligand binding domain comprises an antibody,or a binding portion thereof. In some alternatives, the ligand bindingdomain comprises a single chain variable fragment (scFv), or a bindingportion thereof. In some alternatives, the ligand binding domaincomprises FMC63, or a binding portion thereof. In some alternatives, theligand binding domain is specific for CD19. In some alternatives, thepopulation comprises isolated or enriched CD8+ expressing T-cells in theabsence of, enriched over, substantially depleted of CD4+ T-cells. Insome alternatives, the population comprises isolated or enriched CD4+T-cells in the absence of, enriched over, or substantially depleted ofCD8+ T-cells.

In some alternatives, the adoptive cellular immunotherapy compositionsare useful in the treatment of a disease or cancer. In somealternatives, a composition or product combination for human therapy isprovided, wherein the composition or product combination comprises apharmaceutical excipient and at least one population of geneticallymodified T-cells according to any one or more of the alternative T-cellsor population of genetically modified T-cells prepared or obtained asdescribed herein. In some alternatives of the composition or productcombination for human therapy, the composition or product combinationcomprises a population of genetically modified T-cells, such as T-cellsthat are derived from thymocytes or T-cells that are derived fromengineered precursors, desirably iPS cells, wherein the populationcomprises isolated CD8+ T-cells in the absence of enriched over,substantially depleted of CD4+ T-cells. In some alternatives of thecomposition or product combination for human therapy, the composition orproduct combination comprises a population of genetically modifiedT-cells, such as T-cells that are derived from thymocytes or T-cellsthat are derived from engineered precursors, desirably iPS cells,wherein the population comprises isolated CD4+ T-cells in the absenceof, enriched over, or substantially depleted of CD8+ T-cells. In somealternatives of the composition or product combination for humantherapy, the composition or product combination comprises a combinationof isolated CD8+ T-cells in the absence of, enriched over, orsubstantially depleted of CD4+ T-cells and isolated CD4+ T-cells absenceof, enriched over, or substantially depleted of CD8+ T-cells in a 1:1ratio. In some alternatives, the population of genetically modifiedT-cells are mixed and administered or co-administered as separateformulations to a subject in need thereof in a CD4+ T-cell to CD8+T-cell ratio that is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10or a ratio within a range defined by any two of these ratios. In somealternatives, the population of genetically modified T-cells are mixedand administered or co-administered as separate formulations to asubject in need thereof in a CD8+ T-cell to CD4+ T-cell ratio that is1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a ratio within arange defined by any two of these ratios.

“Pharmaceutical excipient,” or pharmaceutical vehicle as describedherein can refer to a carrier or inert medium used as a solvent in whichthe medicinally active agent or T-cells for treatment is formulated andor administered. Vehicles can include polymeric micelles, liposomes,lipoprotein-based carriers, nano-particle carriers, dendrimers, and/orother vehicles for T-cells that are known to one skilled in the art. Anideal vehicle or excipient can be non-toxic, biocompatible,non-immunogenic, biodegradable, and can avoid recognition by the host'sdefense mechanisms.

In some alternatives, a composition or product combination for humantherapy is provided, wherein the composition or product combinationcomprises a pharmaceutical excipient and at least one population ofgenetically modified T-cells of any of the alternatives describedherein. In some alternatives, the excipients are pharmaceuticalvehicles. In some alternatives, the pharmaceutical vehicles includepharmaceutical compositions.

Acute lymphoblastic leukemia (ALL) or acute lymphoid leukemia is acancer of the white blood cells, characterized by the overproduction ofcancerous, immature white blood cells, also known as lymphoblasts. Inpatients with ALL, lymphoblasts are overproduced in the bone marrow andcontinuously multiply, causing damage and death by inhibiting theproduction of normal cells, such as, for example, red and white bloodcells and platelets, in the bone marrow and by spreading to otherorgans. ALL is most common in childhood with a peak incidence at 2-5years of age, and another peak at an older age.

The symptoms of ALL are indicative of a reduced production of functionalblood cells, because the leukemia wastes the resources of the bonemarrow, which are normally used to produce new, functioning blood cells.Symptoms can include fever, increased risk of infection, increasedtendency to bleed, anemia, tachycardia, fatigue and headache. Between 50to 70% of children and 40-50% of adults who achieve complete remissionafter initial therapy but then suffer a relapse can be able to go into asecond complete remission. Treatment for relapse after a first remissioncan be standard chemotherapy or experimental drugs, or more aggressivetreatments such as stem cell transplants. Treatment for relapse after afirst remission can be standard chemotherapy or experimental drugs, ormore aggressive treatments such as stem cell transplants. However, forothers such as acute myeloid leukemia and ALL the reduced mortality ofthe autogenous relative to allogeneic hematopoietic stem celltransplantation (HSCT) can be outweighed by an increased likelihood ofcancer relapse and related mortality, and therefore the allogeneictreatment can be preferred for those conditions. As such methods areneeded to improve the cells for treatment of leukemia, myeloid leukemiaand ALL.

In some alternatives, a method of treating, inhibiting, or amelioratinga disease, such as a cancer, such as ALL, in a subject in need thereofis provided, wherein the method comprises administering to the subjectat least one composition or product combination of any one or more ofthe alternatives described herein, wherein the composition or productcombination comprises genetically modified T-cells, which have achimeric antigen receptor, wherein the T-cells are transduced,cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells. In somealternatives of the method, the method comprises administering acomposition or product combination, wherein the composition or productcombination comprises a population of genetically modified T-cellscomprising isolated CD8+ T-cells absence of, enriched over, orsubstantially depleted of CD4+ T-cells. In some alternatives of themethod, the method comprises administering a composition or productcombination, wherein the composition or product combination comprises apopulation of genetically modified T-cells comprising isolated CD4+T-cells absence of, enriched over, or substantially depleted of CD8+T-cells. In some alternatives, the method further comprisesadministering a composition or product combination, wherein thecomposition or product combination comprises a population of geneticallymodified T-cells comprising isolated CD4+ T-cells absence of, enrichedover, or substantially depleted of CD8+ T-cells. In some alternatives ofthe method, the method further comprises administering a composition orproduct combination, wherein the composition or product combinationcomprises a population of genetically modified T-cells comprisingisolated CD8+ T-cells absence of, enriched over, or substantiallydepleted of CD4+ T-cells. In some alternatives of the method, the methodfurther comprises administering the composition or product combination,wherein the composition or product combination comprises isolated CD8+T-cells absence of, enriched over, or substantially depleted of CD4+T-cells and isolated CD4+ T-cells absence of, enriched over, orsubstantially depleted of CD8+ T-cells in a 1:1 ratio. In somealternatives, the population of genetically modified T-cells are mixedand administered or co-administered as separate formulations to asubject in need thereof in a CD4+ T-cell to CD8+ T-cell ratio that is1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a ratio within arange defined by any two of these ratios. In some alternatives, thepopulation of genetically modified T-cells are mixed and administered orco-administered as separate formulations to a subject in need thereof ina CD8+ T-cell to CD4+ T-cell ratio that is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10 or a ratio within a range defined by any two ofthese ratios. In some alternatives, the subject is identified orselected to receive an anti-cancer therapy. In some alternatives, themethod further comprises measuring or evaluating an inhibition of adisease. In some alternatives, the method further comprises providingsaid subject an additional anti-cancer therapy before, during, or afteradministration of the composition or product combination of any one ormore of the alternatives described herein. In some alternatives of themethod, the composition or product combination of any one or more of thealternatives described herein, are administered to said subject byadoptive cell transfer. In some alternatives, the composition or productcombination of any one or more of the alternatives described herein, areadministered to said subject after said subject has received anotherform of anti-cancer therapy. In some alternatives, the composition orproduct combination of any one or more of the alternatives describedherein, are administered to said subject after said subject has receivedanother form of anti-cancer therapy. In some alternatives, the subjectis suffering from leukemia. In some alternatives, the subject hasrecurrent and/or refractory CD19+ childhood acute lymphoblastic leukemia(ALL). In some alternatives of the method, the subject has recurrentand/or chemotherapy refractory CD19+ acute lymphoblastic leukemia (ALL).In some alternatives of the method, the subject is suffering from anautoimmune disease. In some alternatives of the method, the subject issuffering from a post-HSCT relapse.

Additional Alternatives Vectors, Cells and Methods of Transducing Cells

The compositions described herein provide for CD4+ and/or CD8+expressing T lymphocytes, such as T-cells that are derived fromthymocytes or T-cells that are derived from engineered precursors,desirably iPS cells. T lymphocytes can be collected in accordance withknown techniques and enriched or depleted by known techniques, such asaffinity binding to antibodies such as flow cytometry and/orimmunomagnetic selection. After enrichment and/or depletion steps, invitro expansion of the desired T lymphocytes can be carried out inaccordance with known techniques (including but not limited to thosedescribed in U.S. Pat. No. 6,040,177 to Riddell et al., hereby expresslyincorporated by reference in its entirety). In some alternatives, theT-cells are autologous T-cells i.e., obtained from the patient to whichthe cells are delivered. In some alternatives, the T cells are precursorT cells. In some alternatives, the precursor T cells are a hematopoieticstem cells (HSC).

For example, the desired T-cell population or subpopulation can beexpanded by adding an initial T lymphocyte population to a culturemedium in vitro, and then adding to the culture medium feeder cells,such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g.,such that the resulting population of cells contains at least 5, 10, 20,or 40 or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T-cells). The non-dividing feedercells can comprise gamma-irradiated PBMC feeder cells. In somealternatives, the PBMC are irradiated with gamma rays at 3000, 3200,3300, 3400, 3500, or 3600 rads to prevent cell division, or within arange of rads defined by any two of the aforementioned rads. The orderof addition of the T-cells and feeder cells to the culture media can bereversed if desired. The culture can be incubated under conditions thatare suitable for the growth of T lymphocytes. For the growth of human Tlymphocytes, for example, the temperature will generally be at least 25degrees Celsius, preferably at least 30 degrees, more preferably 37degrees, or any other temperature within a range defined by any two ofthese temperatures.

The T lymphocytes expanded include CD8+ cytotoxic T lymphocytes (CTL)and CD4+ helper T lymphocytes that can be specific for an antigenpresent on a human tumor or a pathogen and express a chimeric antigenreceptor.

In another alternative, the expansion method or propagation can furthercomprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL)as feeder cells. LCL can be irradiated with gamma rays in the range of6000, 7000, 8000, 9000, or 10,000 rads, or within a range of radsdefined by any two of the aforementioned rads. The LCL feeder cells canbe provided in any suitable amount, such as a ratio of LCL feeder cellsto initial T lymphocytes of at least 10:1.

In another alternative, the expansion method or propagation can furthercomprise adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least 0.5 ng/ml). In anotheralternative, the method of making genetically modified T-cells, whichhave a chimeric antigen receptor method can further comprise addingIL-2, IL-15, and/or IL-21 to the culture medium (e.g., wherein theconcentration of IL-2 is at least 10 units/ml). In another alternative,the method of making genetically modified T-cells, which have a chimericantigen receptor method can further comprise adding IL-7, IL-15, and/orIL21 to the culture medium (e.g., wherein the concentration of IL-2 isat least 10 units/ml). After isolation of T lymphocytes, both cytotoxicand helper T lymphocytes can be sorted into naïve, memory, and effectorT-cell subpopulations either before or after expansion.

CD8+ cells can also be obtained by using standard methods. In somealternatives, CD8+ cells are further sorted into naïve, central memory,and effector memory cells by identifying cell surface antigens that areassociated with each of those types of CD8+ cells. In some alternatives,memory T-cells are present in both CD62L+ and CD62L− subsets of CD8+peripheral blood lymphocytes. PBMC are sorted into CD62L-CD8+ andCD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62Lantibodies. In some alternatives, the expression of phenotypic markersof central memory T_(CM) include CD45RO, CD62L, CCR7, CD28, CD3, and/orCD127 and/or are negative or low for granzyme B and/or CD45RA. In somealternatives, central memory T-cells are CD28+, CD27+, CD45RO+, CD62L+,and/or CD8+ T-cells. In some alternatives, naïve CD8+ T lymphocytes arecharacterized by the expression of phenotypic markers of naïve T-cellsincluding CD62L, CCR7, CD27, CD28, CD3, CD127, and/or CD45RA.

Whether a cell or cell population is positive for or expresses aparticular cell surface marker can be determined by flow cytometry usinga specific antibody that is specific for the surface marker and anisotype matched control antibody. A cell population negative for amarker refers to the absence of significant binding of the cellpopulation with the specific antibody above the isotype control,indicative of a lack of expression of said marker; positive refers touniform binding of the cell population above the isotype controlindicative of the expression of the marker. In some alternatives, adecrease in expression of one or markers refers to loss of 1 log 10 inthe mean fluorescence intensity and/or decrease of percentage of cellsthat exhibit the marker of at least 20% of the cells, 25% of the cells,30% of the cells, 35% of the cells, 40% of the cells, 45% of the cells,50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells,70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells,90% of the cell, 95% of the cells, or 100% of the cells or any % withina range of % s defined by any two of these values when compared to areference cell population. In some alternatives, a cell populationpositive for one or markers refers to a percentage of cells that exhibitthe marker of at least 50% of the cells, 55% of the cells, 60% of thecells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of thecells, 85% of the cells, 90% of the cell, 95% of the cells, or 100% ofthe cells or any % within a range of % s defined by any two of thesevalues when compared to a reference cell population.

CD4+ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In somealternatives, naïve CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+,CD27+, CD28+, and/or CD4+ T-cells. In some alternatives, central memoryCD4+ cells are CD62L+ and/or CD45RO+. In some alternatives, effectorCD4+ cells are CD62L- and/or CD45RO−. In some alternatives, effectorCD4+ cells are CD28+, CD27+ and/or CD62L+.

In some alternatives, populations of CD4+ and CD8+ that are antigenspecific can be obtained by stimulating naïve or antigen specific Tlymphocytes with antigen. For example, antigen-specific T-cell lines orclones can be generated to Cytomegalovirus antigens by isolating T-cellsfrom infected subjects and stimulating the cells in vitro with the sameantigen. Naïve T-cells can also be used. Any number of antigens fromtumor cells can be utilized as targets to elicit T-cell responses. Insome alternatives, the adoptive cellular immunotherapy compositions areuseful in the treatment of a disease or disorder including a solidtumor, hematologic malignancy, breast cancer or melanoma.

Modification of T Lymphocyte Populations

In some alternatives it can be desired to introduce functional genesinto the T-cells to be used in immunotherapy in accordance with thepresent disclosure. For example, the introduced gene or genes canimprove the efficacy of therapy by promoting the viability and/orfunction of transferred T-cells; or they can provide a genetic marker topermit selection and/or evaluation of in vivo survival or migration; orthey can incorporate functions that improve the safety of immunotherapy,for example, by making the cell susceptible to negative selection invivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6(1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); seealso the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton etal. describing the use of bifunctional selectable fusion genes derivedfrom fusing a dominant positive selectable marker with a negativeselectable marker, both references hereby expressly incorporated byreference in their entireties. This can be carried out in accordancewith known techniques (see, e.g., U.S. Pat. No. 6,040,177 to Riddell etal. at columns 14-17, hereby expressly incorporated by reference in itsentirety) or variations thereof that will be apparent to those skilledin the art based upon the present disclosure. In some alternatives, theT cells are precursor T cells. In some alternatives, the precursor Tcells are a hematopoietic stem cells (HSC).

In some alternatives, T-cells are modified with chimeric receptors, asdescribed herein. In some alternatives, the T-cells are obtained fromthe subject to be treated, in other alternatives, the lymphocytes areobtained from allogeneic human donors, preferably healthy human donors.In some alternatives, the T cells are precursor T cells. In somealternatives, the precursor T cells are a hematopoietic stem cells(HSC).

In some alternatives, chimeric receptors comprise a ligand bindingdomain that specifically binds to surface molecule on a cell, apolypeptide spacer region, a transmembrane domain and an intracellularsignaling domain as described herein. In some alternatives, the ligandbinding domain is a single-chain antibody fragment (scFv) that isderived from the variable heavy (VH) and variable light (VL) chains of amonoclonal antibody (mAb). Co-stimulatory signals can also be providedthrough the chimeric receptor by fusing the co-stimulatory domain ofCD28 and/or 4-1BB to the CD3ξ chain. Chimeric receptors are specific forcell surface molecules independent from HLA, thus overcoming thelimitations of TCR-recognition including HLA-restriction and low levelsof HLA-expression on tumor cells.

In some alternatives, the same or a different chimeric receptor can beintroduced into each of population of CD4+ and CD8+ T lymphocytes. Insome alternatives, the chimeric receptor in each of these populationshas a ligand binding domain that specifically binds to the same ligandon the cell. The cellular signaling modules can differ. In somealternatives, the intracellular signaling domain of the CD8+ cytotoxicT-cells is the same as the intracellular signaling domain of the CD4+helper T-cells. In other alternatives, the intracellular signalingdomain of the CD8+ cytotoxic T-cells is different than the intracellularsignaling domain of the CD4+ helper T-cells. In some alternatives, ananti-CD19 CAR is introduced into one population of lymphocytes and anEGFR specific CAR is introduced into another population of lymphocytes.

In some alternatives each of the CD4 or CD8 T lymphocytes are sorted into naïve, central memory, effector memory or effector cells prior totransduction, as described herein. In some alternatives, each of the CD4or CD8 T lymphocytes are sorted into naïve, central memory, effectormemory, or effector cells after transduction.

Various transduction techniques have been developed, which utilizerecombinant infectious virus particles for gene delivery. Thisrepresents a currently preferred approach to the transduction of Tlymphocytes described herein. The viral vectors, which have been used inthis way include virus vectors derived from simian virus 40,adenoviruses, adeno-associated virus (AAV), lentiviral vectors, andretroviruses. Thus, gene transfer and expression methods are numerousbut essentially function to introduce and express genetic material inmammalian cells. Several of the above techniques have been used totransduce hematopoietic or lymphoid cells, including calcium phosphatetransfection, protoplast fusion, electroporation, and infection withrecombinant adenovirus, adeno-associated virus and retrovirus vectors.Primary T lymphocytes have been successfully transduced byelectroporation and by retroviral or lentiviral infection.

Retroviral and lentiviral vectors provide a highly efficient method forgene transfer into eukaryotic cells. Moreover, retroviral or lentiviralintegration takes place in a controlled fashion and results in thestable integration of one or a few copies of the new genetic informationper cell.

In some alternatives it can be useful to include in the T-cells apositive marker that enables the selection of cells of the negativeselectable phenotype in vitro. The positive selectable marker can be agene that upon being introduced into the host cell expresses a dominantphenotype permitting positive selection of cells carrying the gene.Genes of this type are known in the art, and include, inter alia,hygromycin-B phosphotransferase gene (hph) which confers resistance tohygromycin B, the amino glycoside phosphotransferase gene (neo or aph)from Tn5 which codes for resistance to the antibiotic G418, thedihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA),and the multi-drug resistance (MDR) gene. In some alternatives, thepositive marker is an EGFR truncated protein for cell selection.

A variety of methods can be employed for transducing T lymphocytes, asis well known in the art. In some alternatives, transduction is carriedout using lentiviral vectors. In some alternatives, CD4+ and CD8+ cellseach can separately be modified with an expression vector encoding achimeric receptor to form defined populations. In some alternatives,these cells are then further sorted into subpopulations of naïve,central memory and effector cells as described above by sorting for cellsurface antigens unique to each of those cell populations.

In some alternatives, CD4+ and CD8+ cells that proliferate in responseto antigen or tumor targets are selected. For example, CD4+ cells thatproliferate vigorously when stimulated with antigen or tumor targets ascompared to sham transduced cells, or CD8+ transduced cells areselected. In some alternatives, CD4+ and CD8+ cells are selected thatare cytotoxic for antigen bearing cells. In some alternatives, CD4+ areexpected to be weakly cytotoxic as compared to CD8+ cells.

In another alternative, transduced lymphocytes, such as CD8+ centralmemory cells, are selected that provide for cell killing in vivo usingan animal model established for the particular type of cancer. Suchanimal models are known to those of skill in the art and exclude humanbeings. As described herein, not all chimeric receptor constructstransduced into lymphocytes confer the ability to kill tumor cells invivo despite the ability to become activated and kill cells in vitro.

The disclosure contemplates that combinations of CD4+ and CD8+ T-cellswill be utilized in compositions. In one alternative, combinations ofchimeric receptor transduced CD4+ cells can be combined with chimericreceptor transduced CD8+ cells of the same ligand specificity orcombined with CD8+ T-cells that are specific for a distinct tumorligand. In other alternatives, chimeric receptor transduced CD8+ cellsare combined with chimeric receptor transduced CD4+ cells specific for adifferent ligand expressed on the tumor. In yet another alternative,chimeric receptor modified CD4+ and CD8+ cells are combined. In somealternatives CD8+ and CD4+ cells can be combined in different ratios forexample, a 1:1 ratio of CD8+ and CD4+, a ratio of 10:1 of CD8+ to CD4+,or a ratio of 100:1 of CD8+ to CD4+ or any other ratio within a rangedefined by any two of the aforementioned ratio values. In somealternatives, the combined population is tested for cell proliferationin vitro and/or in vivo, and the ratio of cells that provides forproliferation of cells is selected.

As described herein, the disclosure contemplates that CD4+ and CD8+cells can be further separated into subpopulations, such as naïve,central memory, and effector memory cell populations. As describedherein, in some alternatives, naïve CD4+ cells are CD45RO−, CD45RA+,CD62L+, CD28+, CD27+, and/or CD4+ positive T-cells. In somealternatives, central memory CD4+ cells are CD62L positive and/or CD45ROpositive. In some alternatives, effector CD4+ cells are CD62L negativeand/or CD45RO positive. Each of these populations can be independentlymodified with a chimeric receptor. In some alternatives, central memoryCD4+ cells are CD62L+, CD28+ and/or CD27+.

After transduction and/or selection for chimeric receptor bearing cells,the cell populations are preferably expanded in vitro until a sufficientnumber of cells are obtained to provide for at least one infusion into ahuman subject, typically around 10⁴ cells/kg to 10⁹ cells/kg. In somealternatives, the transduced cells are cultured in the presence ofantigen bearing cells, anti CD3, anti CD28, IL 2, IL-7, IL 15, and/orIL-21 and/or combinations thereof. In some alternatives, the T-cells arestimulated in the presence of an antibody-bound support, such as a beador particle. In some alternatives, the T-cells are stimulated with anantibody-bound support, wherein the antibody-bound support comprisesanti-TCR, anti-CD2, anti-CD3, anti-CD4 and/or anti-CD28 antibodies. Insome alternatives, the T-cells are stimulated with an antibody-boundsupport, wherein the antibody-bound support comprises anti-CD3 and/oranti-CD28 antibodies.

Each of the subpopulations of CD4+ and CD8+ cells can be combined withone another. In a specific alternative, modified naïve or central memoryCD4+ cells are combined with modified central memory CD8+ T-cells toprovide a synergistic cytotoxic effect on an antigen bearing cells, suchas a B-cell comprising CD19 on the cell surface.

Compositions

The disclosure provides for an adoptive cellular immunotherapycomposition comprising a genetically modified T lymphocyte cellpreparation as described herein.

In some alternatives, the T lymphocyte cell preparation comprises CD4+T-cells that have a chimeric receptor comprising an extracellularantibody variable domain specific for a ligand associated with thedisease or disorder, a customizable spacer region, a transmembranedomain, and an intracellular signaling domain of a T-cell receptor orother receptors as described herein. In other alternatives, an adoptivecellular immunotherapy composition further comprises a chimeric receptormodified tumor-specific CD8+ cytotoxic T lymphocyte cell preparationthat provides a cellular immune response, wherein the cytotoxic Tlymphocyte cell preparation comprises CD8+ T-cells that have a chimericreceptor comprising an extracellular single chain antibody specific fora ligand associated with the disease or disorder, a customizable spacerregion, a transmembrane domain, and an intracellular signaling domain ofa T-cell receptor, as described herein.

In some alternatives, an adoptive cellular immunotherapy compositioncomprises a chimeric receptor modified tumor-specific CD8+ cytotoxic Tlymphocyte cell preparation that provides a cellular immune response,wherein the cytotoxic T lymphocyte cell preparation comprises CD8+T-cells that have a chimeric receptor comprising an extracellular singlechain antibody specific for a ligand associated with the disease ordisorder, a customizable spacer region, a transmembrane domain, and anintracellular signaling domain of a T-cell receptor, in combination withan antigen-reactive chimeric receptor modified naïve CD4+ T helper cellderived from CD45RO− CD62L+ and/or CD4+ T-cells, and a pharmaceuticallyacceptable carrier.

In other alternatives, an adoptive cellular immunotherapy compositioncomprises an antigen specific CD8+ cytotoxic T lymphocyte cellpreparation that provides a cellular immune response derived from thepatient combined with an antigen-reactive chimeric receptor modifiednaïve CD4+ T helper cell that augments the CD8+ immune response, whereinthe helper T lymphocyte cell preparation comprises CD4+ T-cells thathave a chimeric receptor comprising an extracellular antibody variabledomain specific for the antigen associated with the disease or disorder,a customizable spacer region, a transmembrane domain, and anintracellular signaling domain of a T-cell receptor.

In a further alternative, an adoptive cellular immunotherapy compositioncomprises an antigen-reactive chimeric receptor modified naïve CD4+ Thelper cell that augments the CD8+ immune response, wherein the helper Tlymphocyte cell preparation comprises CD4+ T-cells that have a chimericreceptor comprising an extracellular antibody variable domain specificfor a ligand associated with a disease or disorder, a customizablespacer region, a transmembrane domain, and an intracellular signalingdomain of a T-cell receptor.

In some alternatives, the CD4+ T helper lymphocyte cell is selected fromthe group consisting of naïve CD4+ T-cells, central memory CD4+ T-cells,effector memory CD4+ T-cells, and bulk CD4+ T-cells. In somealternatives, CD4+ helper lymphocyte cell is a naïve CD4+ T-cell,wherein the naïve CD4+ T-cell comprises a CD45RO−, CD45RA+, CD62L+CD28+,CD27+, and/or CD4+. In some alternatives, the CD8+ T cytotoxiclymphocyte cell is selected from the group consisting of naïve CD8+T-cells, central memory CD8+ T-cells, effector memory CD8+ T-cells andbulk CD8+ T-cells. In some alternatives, the CD8+ cytotoxic T lymphocytecell is a central memory T-cell wherein the central memory T-cellcomprises a CD45RO+, CD62L+, CD27+, CD28+ and/or CD8+. In yet otheralternatives, the CD8+ cytotoxic T lymphocyte cell is a central memoryT-cell and the CD4+ helper T lymphocyte cell is a naïve or centralmemory CD4+ T-cell.

The disclosure provides methods of making adoptive immunotherapycompositions and uses or methods of using these compositions forperforming cellular immunotherapy in a subject having a disease ordisorder. Proliferation and persistence of the chimeric receptormodified T-cells can be determined by using an animal model of thedisease or disorder and administering the cells and determiningpersistence and/or proliferative capacity of the transferred cells. Inother alternatives, proliferation and activation can be tested in vitroby going through multiple cycles of activation with antigen bearingcells.

In some alternatives, a method of manufacturing the compositionscomprises obtaining a modified naïve CD4+ T helper cell, wherein themodified helper T lymphocyte cell preparation comprises CD4+ T-cellsthat have a chimeric receptor comprising a ligand binding domainspecific for a tumor cell surface molecule, a customized spacer domain,a transmembrane domain, and an intracellular signaling domain asdescribed herein.

In another alternative, a method further comprises obtaining a modifiedCD8+ cytotoxic T cell, wherein the modified cytotoxic T lymphocyte cellpreparation comprises CD8+ cells that have a chimeric receptorcomprising a ligand binding domain specific for a tumor cell surfacemolecule, a customized spacer domain, a transmembrane domain, and anintracellular signaling domain as described herein.

In another alternative, a method comprises obtaining a modified CD8+cytotoxic T-cell, wherein the modified cytotoxic T lymphocyte cellpreparation comprises CD8+ T-cells that have a chimeric receptorcomprising a ligand binding domain specific for a tumor cell surfacemolecule, a customized spacer domain, a transmembrane domain, and anintracellular signaling domain, as described herein, and furthercomprising combining the modified CD8+ cytotoxic T-cells with a CD4+helper cell lymphocyte cell preparation.

The preparation of the CD4+ and CD8+ cells that are modified with achimeric receptor has been described above, as well as, in the examples.Antigen specific T lymphocytes can be obtained from a patient having thedisease or disorder or can be prepared by in vitro stimulation of Tlymphocytes in the presence of antigen. Subpopulations of CD4+ and CD8+T lymphocytes that are not selected for antigen specificity can also beisolated as described herein and combined in the methods ofmanufacturing. In some alternatives, the combination of cell populationscan be evaluated for uniformity of cell surface makers, the ability toproliferate through at least two generations, to have a uniform celldifferentiation status. Quality control can be performed by co-culturinga cell line expressing the target ligand with chimeric receptor modifiedT-cells to determine if the chimeric receptor modified T-cells recognizethe cell line using cytotoxicity, proliferation, or cytokine productionassays that are known in the field. Cell differentiation status and cellsurface markers on the chimeric receptor modified T-cells can bedetermined by flow cytometry. In some alternatives, the markers and celldifferentiation status on the CD8+ cells include CD3, CD8, CD62L, CD28,CD27, CD69, CD25, PD-1, CTLA-4, CD45RO, and/or CD45RA. In somealternatives, the markers and the cell differentiation status on theCD4+ cells include CD3, CD4, CD62L, CD28, CD27, CD69, CD25, PD-1, CTLA-4CD45RO, and/or CD45RA. In some alternatives, the markers include CD45RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. Insome alternatives, the markers include CD27, CD28 and/or CD62L.

Some alternatives relate to approaches for treating, inhibiting, orameliorating a disease, such as cancer, such as ALL in a subject in needthereof, including methods of inhibiting or delaying progression and/ormetastasis of a cancer, methods of inhibiting or reducing the presenceof a tumor or cancer cell, and/or a methods of inhibiting or reducing atarget population of CD19 expressing cells in a patient in need thereof.Such methods involve administering to a subject or a patient in needthereof a genetically modified cytotoxic T lymphocyte cell preparationthat provides a cellular immune response, wherein the cytotoxic Tlymphocyte cell preparation comprises CD8+ T-cells that have a chimericreceptor comprising a polynucleotide coding for a ligand binding domain,wherein the ligand is a tumor specific antigen, or any other moleculeexpressed on a target-cell population (e.g. CD19) that is suitable tomediate recognition and elimination by a lymphocyte; a polynucleotidecoding for a polypeptide spacer wherein the polypeptide spacer is of acustomized length, wherein the spacer provides for enhanced T-cellproliferation and/or cytokine production as compared to a referencechimeric receptor; a polynucleotide coding for a transmembrane domain;and a polynucleotide coding for one or more intracellular signalingdomains.

The disclosure also provides methods of performing cellularimmunotherapy in a subject having a disease or disorder such as cancer,such as ALL comprising: administering a composition of lymphocytesexpressing a chimeric receptor as described herein. In otheralternatives, a method comprises administering to the subject agenetically modified cytotoxic T lymphocyte cell preparation thatprovides a cellular immune response, wherein the cytotoxic T lymphocytecell preparation comprises CD8+ T-cells that have a chimeric receptorcomprising a ligand binding domain specific for a cell surface molecule,a customized spacer domain, a transmembrane domain, and an intracellularsignaling domain as described herein, and a genetically modified helperT lymphocyte cell preparation that elicits direct recognition andaugments the genetically modified cytotoxic T lymphocyte cellpreparations ability to mediate a cellular immune response, wherein thehelper T lymphocyte cell preparation comprises CD4+ T-cells that have achimeric receptor comprising a ligand binding domain specific for a cellsurface molecule, a customized spacer domain, a transmembrane domain,and an intracellular signaling domain, as described herein.

While not limiting the scope of the disclosure, it is believed byselecting the chimeric receptor modified T-cell population that canpersist and proliferate in vivo prior to administration can result inthe ability to use a lower dose of T-cells and provide more uniformtherapeutic activity. In some alternatives, the dose of T-cells can bereduced at least 10%, 20%, or 30% or greater. Reduction in the dose ofT-cells can be beneficial to reduce the risk or tumor lysis syndrome andcytokine storm.

In another alternative, a method of performing cellular immunotherapy insubject having a disease or disorder comprises: administering to thesubject a genetically modified helper T lymphocyte cell preparation,wherein the modified helper T lymphocyte cell preparation comprises CD4+T-cells that have a chimeric receptor comprising a ligand binding domainspecific for a tumor cell surface molecule, a customized spacer domain,a transmembrane domain, and an intracellular signaling domain asdescribed herein. In some alternatives, the method further comprisesadministering to the subject a genetically modified cytotoxic Tlymphocyte cell preparation, wherein the modified cytotoxic T lymphocytecell preparation comprises CD4+ cells that have a chimeric receptorcomprising a ligand binding domain specific for a tumor cell surfacemolecule, a customized spacer domain, a transmembrane domain, and anintracellular signaling domain as described herein.

Another alternative describes a method of performing cellularimmunotherapy in a subject having a disease or disorder comprising:analyzing a biological sample of the subject for the presence of atarget molecule (e.g. CD19) associated with the disease or disorder andadministering the adoptive immunotherapy compositions described herein,wherein the chimeric receptor specifically binds to the target molecule(CD19).

In some alternatives, the CD4+ T helper lymphocyte cell is selectedprior to introduction of the chimeric receptor from the group consistingof naïve CD4+ T-cells, central memory CD4+ T-cells, effector memory CD4+T-cells and bulk CD4+ T-cells. In a specific alternative, CD4+ helperlymphocyte cell is a naïve CD4+ T-cell, wherein the naïve CD4+ T-cellcomprises a CD45RO−, CD45RA+, CD28+, CD27+, CD62L+ and/or CD4+. In yetother alternatives, the CD8+ T cytotoxic lymphocyte cell is selectedprior to introduction of the chimeric receptor from the group consistingof naïve CD8+ T-cells, central memory CD8+ T-cells, effector memory CD8+T-cells and bulk CD8+ T-cells. In a specific alternative, the CD8+cytotoxic T lymphocyte cell is a central memory T-cell wherein thecentral memory T-cell comprises a CD45RO+, CD62L+, CD28+, CD28+, and/orCD8+. In a specific alternative, the CD8+ cytotoxic T lymphocyte cell isa central memory T-cell and the CD4+ helper T lymphocyte cell is a naïveCD4+ T-cell.

In some alternatives, the CD8+ T-cell and the CD4+ T-cell are bothgenetically modified with a chimeric receptor comprising an antibodyheavy chain domain that specifically binds a cell surface molecule. Inother alternatives, the intracellular signaling domain of the CD8cytotoxic T-cells is the same as the intracellular signaling domain ofthe CD4 helper T-cells. In yet other alternatives, the intracellularsignaling domain of the CD8 cytotoxic T-cells is different than theintracellular signaling domain of the CD4 helper T-cells.

The subjects that can be administered the alternatives described hereininclude humans, other primates, such as monkeys and apes, companionanimals, such as dogs, cats, and horses, and domestic animals such aspigs, goats, sheep, and cattle. The subjects can be male or female andcan be any suitable age, including infant, juvenile, adolescent, adult,and geriatric subjects. The methods are useful in the treatment of, forexample, CD19 bearing cancer or cells.

Cells prepared as described above can be utilized in methods andcompositions for adoptive immunotherapy in accordance with knowntechniques, or variations thereof that will be apparent to those skilledin the art based on the instant disclosure.

In some alternatives, the cells are formulated by first harvesting themfrom their culture medium, and then washing and concentrating the cellsin a medium and container system suitable for administration (a“pharmaceutically acceptable” carrier) in a treatment-effective amount.Suitable infusion medium can be any isotonic medium formulation,typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter),but also 5% dextrose in water or Ringer's lactate can be utilized. Theinfusion medium can be supplemented with fetal calf serum. Cells arethen treated with cytokines to produce a transduced, cytokine-stimulatedpopulation of CD8+ T-cells and/or CD4+ T-cells with an increased orimproved engraftment fitness as compared to non-cytokine stimulatedpopulations of CD8+ T-cells and/or CD4+ T-cells. In order to generatecells with the desired improvement in engraftment fitness, the cytokinesare desirably added in vitro during the propagation of the T-cells. Assuch, separated CD4+ and CD8+ T-cells are cytokine stimulated by theaddition of cytokines during the propagation of the genetically modifiedT-cells. In some alternatives, the CD4+ T cells are stimulated with atleast one cytokine, wherein the at least one cytokine comprises IL/7,IL-15 and/or IL-21. In some alternatives, the CD8+ T cells arestimulated with at least one cytokine, wherein the at least one cytokinecomprises IL-2, IL-15 and/or IL-21.

A treatment or inhibitory effective amount of cells in the compositionis at least 2 cell subsets (for example, 1 CD8+ central memory T-cellsubset and 1 CD4+ helper T-cell subset) or is more typically greaterthan 10² cells, and up to 10⁶, up to and including 10⁸ or 10⁹ cells andcan be more than 10¹⁰ cells or any other value in a range defined by anyof these two values. The number of cells will depend upon the ultimateuse for which the composition is intended as will the type of cellsincluded therein. For example, if cells that are specific for aparticular antigen are desired, then the population will contain greaterthan 70%, generally greater than 80%, 85% and/or 90-95% of such cells.For uses provided herein, the cells are generally in a volume of a literor less, can be 500 mls or less, even 250 mls or 100 mls or less, or anyother value within a range defined by any two of these values. Hence thedensity of the desired cells is typically greater than 10⁴ cells/ml andgenerally is greater than 10⁷ cells/ml, generally 10⁸ cells/ml orgreater. The clinically relevant number of immune cells can beapportioned into multiple infusions that cumulatively equal or exceed10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ or 10¹¹ cells or any other amount within arange defined by any of these two values.

In some alternatives, the lymphocytes of the invention can be used toconfer immunity to individuals. By “immunity” is meant a lessening ofone or more physical symptoms associated with a response to infection bya pathogen, or to a tumor, to which the lymphocyte response is directed.The amount of cells administered is usually in the range present innormal individuals with immunity to the pathogen. Thus, the cells areusually administered by infusion, with each infusion in a range of from2 cells, up to at least 10⁶ to 3×10¹⁰ cells, preferably in the range ofat least 10⁷ to 10⁹ cells. The T-cells can be administered by a singleinfusion, or by multiple infusions over a range of time. However, sincedifferent individuals are expected to vary in responsiveness, the typeand amount of cells infused, as well as the number of infusions and thetime range over which multiple infusions are given are determined by theattending physician, and can be determined by routine examination. Thegeneration of sufficient levels of T lymphocytes (including cytotoxic Tlymphocytes and/or helper T lymphocytes) is readily achievable using therapid expansion method of the present invention, as exemplified herein.See, e.g., U.S. Pat. No. 6,040,177 to Riddell et al. at column 17,hereby expressly incorporated by reference in its entirety.

In some alternatives, the compositions, as described herein areadministered intravenously, intraperitoneally, intratumorly, into thebone marrow, into the lymph node, and/or into cerebrospinal fluid.

In some alternatives, the compositions, as described herein areadministered with chemotherapeutic agents and/or immunosuppressants. Inan alternative, a patient is first administered a chemotherapeutic agentthat inhibits or destroys other immune cells followed by thecompositions described herein. In some cases, chemotherapy can beavoided entirely.

Administration to Patients of ALL

CD4 and CD8 T cell subsets were immunomagnetically isolated fromapheresis products obtained from the research participant. Followinganti-CD3×CD28 bead stimulation, T cell lines were transduced with a SINlentiviral vector that directs the co-expression of theFMC63scFv:IgG4hinge: CD28tm:4-1BB:ξ CAR and the selection and trackingsuicide construct EGFRt. Transduced cells were propagated usingrecombinant human cytokines to numbers suitable for clinical use over10-20 days during which time they were subjected to EGFRt immunomagneticpositive selection. Shortly following lymphodepleting chemotherapy,cryopreserved CD4/EGFRt+ and CD8/EGFRt+ T cell products were thawed andinfused at the bedside such that patients received a 1:1 ratio of EGFRt+CD4 and CD8 T cells at the indicated protocol-prescribed dose level(1=0.5×10⁶/kg; 2=1×10⁶/kg; 3=5×10⁶/kg). In some alternatives, theenriched CD4+ expressing T-cells were contacted, for example propagated,in 5 ng/mL recombinant human IL-7 (rhIL-7) and/or 0.5 ng/mL recombinanthuman IL-15 (rhIL-15). In some alternatives, the enriched CD8+expressing T-cells were contacted, for example propagated, in 50 U/mLrecombinant human IL-2 (rhIL-2) and/or 0.5 ng/mL recombinant human IL-15(rhIL-15).

All enrolled subjects (n=16) had cell products that met dosingspecification and had been cleared for infusion. 13 subjects (6 m-3 yrs/p HSCT) had been treated at varying dose levels (range of0.5×10⁶/kg-5×10⁶/kg). The infusions were well tolerated with only 1AE>grade 2 (grade 3 anaphylaxis related to the DMSO). 12 out of 13subjects had responses for an ORR of 92%. 11 out of 13 subjects achievedMRD negative CRs (85%). Subjects with higher disease burden had higherpeak PB CAR T cell levels compared to those with MRD negative marrows(62.7% v 19.6%). Accumulation with expansion of CAR T cells in bonemarrow (n=12), peripheral blood (n=12) and CSF was observed. The averageduration of persistence for the 5 subjects who lost their T cell graftswas 63 days (range of 42-150 d).

Each of the twelve responding subjects developed some degree of CRS withfever and hypotension as the hallmark symptoms. Two subjects, S02 andS09, required immunomodulatory treatment for sCRS (tocilizumab for two,and addition of dexamethasone in one). Four of 13 of these patientsdeveloped encephalopathy (2 grade 1, 1 grade 3 and 1 grade 4). The grade4 encephalopathy was a DLT, and accompanied by seizures and abnormal MRIfindings similar to PRES. MRI findings were normalized by 9 weeks posttherapy, but the subject, at the time of last assessment, continued tohave seizures, 6 months from therapy.

As shown, infusions of prescribed dose levels of defined compositionCD4:CD8 CD19CAR/EGFRt+ T cells/kg produced encouraging rates ofMRD-negative CRs in pediatric and young adult ALL patients who havesuffered a post-HSCT relapse. As such, it is feasible to generatedonor-derived products from each of the sixteen enrolled patients. Theexpected toxicities for the 13 evaluable patients include CRS with ˜30%ICU admission rate and encephalopathy with severity ranging from mild tosevere. MRD negative responses have been seen at all dose levels,although early data suggests enhanced persistence at escalating doses.

Although one patient developed acute GVHD post T cell therapy, thepreliminary assessment suggested that CAR T cells were not mediators ofthis response. Interestingly, prednisone did not affect persistence ofthe T cell graft. As such, the cells that maintained the markers CD28,CD27, and CD62L showed high persistence in comparison to cells that werenot treated with the defined cytokine mixtures and lack the markers forengraftment fitness, which are the T-cells that are used for the currentmethod of treatment.

Shown in FIG. 18, is a flow diagram depicting the development history ofa process used to manufacture the genetically modified T-cell comprisinga chimeric antigen receptor. As indicated, the current manufacturingprocedure does not have the Ficoll processing, whereas initialexperimental processes included a Ficoll processing step. The initialvariations of cell concentrations were also tested as shown in the flowdiagram of FIG. 19. Cells were also expanded in the presence ofcytokines (FIG. 20).

As shown in FIG. 20, donor sample PD0064: PD0063 CD8+ enriched T-cellswere used to test starting cell density in a T25 flask on initiation.Both cells (from PD0064, PD0063) were tested under normal PLAT-02culture conditions, IL-2 (50 U/ml)+IL-15 (0.5 ng/mL). Cells from donorsample PD0080, were used in a study to test the expansion of cells on Txday, by adding media and cytokines for a final cell concentration of to0.7×10̂6 cell/mL (“early expansion”). By “Tx” it is meant throughout torefer to Day +1, based on the day of stimulation beads being defined asDay +0. PD0063 CD8+ cells were used as the enriched starting materialThe same cytokine conditions were used as with the PD0064:PD0063 CD8+enriched cells. The experiment tested two cell densities both showinggreater viability and expansion when the cells were volumed up early inthe presence of the cytokines. In this context “volume up” refers to thevolume of media/cytokine addition for initial feeding following Tx wasgreater in the “early expansion” conditions, leading to decreased celldensity at this point in culture for “early expansion” conditions.

As shown in FIG. 21, are the growth of cell samples PD104 and PD0116 andthe comparison of cell growth under experimental conditions. PD0104:PD0063 CD8+ and CD4+ enriched cells were used to repeat previousexperiments to test the expansion of cells on Tx day to 0.7×10⁶ cell/mL(“early expansion”), with the following cytokines and respectiveconcentrations being added: IL-2 (50 U/ml)+IL-15 (0.5 ng/mL) for CD8+ T−cells and IL-7 (5 ng/mL)+IL-15 (0.5 ng/mL) for CD4+ T-cells. Growth andviability were increased dramatically. These curves mimic what was seenin clinical products as far as timing of growth/beadremoval/selection/cryopreservation.

The PD0116: PD0046 CD8+ and CD4+ enriched cells were used to repeat theexperiment to test the expansion of cells on Tx day to 0.7×10̂6 cell/mL(“early expansion”). Normal PLAT-02 culture conditions, IL-2 (50U/ml)+IL-15 (0.5 ng/mL) for CD8 cells and IL-7 (5 ng/mL)+IL-15 (0.5ng/mL) for CD4's. PD116 was initiated at 180×10̂6 cells in a V-197 bagand magnetic enrichment and spinoculation were performed in a transferpack. As shown, from these conditions, growth and viability wereincreased dramatically. These curves mimic what was seen in clinicalproducts as far as timing of growth, bead removal, selection, andcryopreservation of T-cells.

PD0116: PD0046 CD8+ and CD4+ enriched cells were also used to repeat anexperiment to test the expansion of cells on Tx day by the addition ofmedia and cytokines for a final volume of to 0.7×10⁶ cell/mL (“earlyexpansion”). Normal PLAT-02 culture conditions in which additions ofIL-2 (50 U/ml)+IL-15 (0.5 ng/mL) for CD8 cells and IL-7 (5 ng/mL)+IL-15(0.5 ng/mL) for CD4's was performed. PD116 was initiated at 180×10⁶cells in a V-197 bag and magnetic enrichment and spinoculation wereperformed in a transfer pack. This was the full scale data used totransition to an updated manufacturing process. As shown in the FIG. 21,growth expansion was increased for CD8+ cells in comparison to the CD4+cells. The cytokines used for the CD4_(—) and the CD8+ cells and thevariations of the combinations of the cytokines used are depicted in aflow chart as shown in FIG. 22.

Various cytokine conditions were also tested on the CD8+ and the CD4+cells from donors. As shown in FIG. 23 panels A-F, samples from PD0047and PD0040 were tested with IL2/IL15, IL7/IL15, IL2/IL7/IL15, IL2 pulsewith IL7/IL15, IL2 only, IL2 pulse with IL7/IL21 and IL2/IL15. Thisincluded incubation with IL-2 alone (pulse) for 5 days, followed by theaddition of IL-7/IL-21, which then were present throughout the remainderof the culture, incubation with IL-2 alone (pulse) for 5 days, followedby the addition of IL-7/IL-21 and IL2/IL15, which then were presentthroughout the remainder of the culture. PD0040: PD0038 CD8+ enrichedT-cells were used to test various cytokine cocktails. Concentrations foreach condition were: IL-2 (50 U/mL), IL-7 (5 ng/mL), IL-15 (0.5 ng/mL).Condition #4 was an IL-2 pulse for 5 days followed by IL-7/IL-15throughout the remainder of the culture. Fold growth and TNC wereextrapolated from small scale experiment. Currently used IL-2/IL-15combination showed best expansion. In this study, the IL-2/IL-15combination was demonstrated to result in the best expansion.

As shown, the PD0047: PD0038 CD8+ enriched cells were used to testvarious cytokine cocktails. The same cytokine cocktails were testedusing “low dose” IL-2 at 10 U/mL final. Concentrations of the individualcytokines used in the various combinations were: IL-2 (50 U/mL) and (10U/mL) as stated in the legend, IL-7 (5 ng/mL), IL-15 (0.5 ng/mL), IL-21(5 ng/mL). Pulse conditions were an IL-2 pulse for 5 days followed byIL-7/IL-21 throughout the remainder of the culture. Fold growth and TNCare extrapolated from a small scale experiment. The IL-2/IL-15combination showed the best expansion and was used for followingexperimentations. The “normal” dose of IL-2 showed best growth. Panel Ashows results from incubation of PD0047 cells at day 15 (D15) and thecomparison of growth with various cytokines and the percent positivecells (CD3+, CD62L+, CD3+/CD62L+/CD45RO−, CD28+, CDD45RA+/CD4RO−,CD45RA+/CD45RO and CD45RA−/CD45RO+), following incubation with thevarious cytokine conditions.

Panel B shows results following incubation of PD0047 cells at day 21(D21) and the comparison of growth with various cytokines and thepercent positive cells as indicated above. Panel C shows PD0040 D21 andthe comparison of growth with various cytokines, in which the total cellnumber is examined after 22 days of growth in culture. Panel D showsPD0040 D21 and the comparison of growth with various cytokines, asindicated the CD3+ cell growth is examined after 22 day of culturegrowth. Panel E shows PD0047 and the comparison of growth with variouscytokines, in which the total cell number is examined after 22 days ofgrowth in culture. Panel F shows PD0047 and the comparison of growthwith various cytokines, as indicated the CD3+ cell growth is examinedafter 22 day of culture growth. As shown for PD0040: PD0038 CD8+enriched cells, were used to test various cytokine cocktails.Concentrations for each condition were: IL-2 (50 U/mL), IL-7 (5 ng/mL),IL-15 (0.5 ng/mL). Condition #4 was an IL-2 pulse for 5 days followed byIL-7/IL-15 throughout the remainder of the culture. Fold growth and TNCwere extrapolated from small scale experiments. IL-2/IL-15 combinationshowed the best expansion and was used for future experiments.Additionally, PD0047: PD0038 CD8+ enriched cells were used to testvarious cytokine cocktails. The same cytokine cocktails were testedusing “low dose” IL-2 at 10 U/mL final. Concentrations for eachcondition were: IL-2 (50 U/mL) and (10 U/mL) stated in legend, IL-7 (5ng/mL), IL-15 (0.5 ng/mL), IL-21 (5 ng/mL). Pulse conditions were anIL-2 pulse for 5 days followed by IL-7/IL-21 throughout the remainder ofthe culture. Fold growth and TNC were then extrapolated from small scaleexperiments. The IL-2/IL-15 cytokine combination used in the studydescribed above showed the best expansion. The “normal” dose of IL-2also showed the best growth. Flow cytometry for phenotypic growth isavailable and indicated in further experiments herein.

Shown in FIG. 24, are results from a series of experiments to test thedifferent combination of cytokines PD0051: CD8+ cells from two differentdonors were used to test the “normal” cytokine conditions using IL-2 (50U/mL) and IL-15 (0.5 ng/mL) were used. Pulse conditions were an IL-2pulse for 5 days followed by IL-7 (5 ng/mL), IL-21 (5 ng/mL), throughoutthe remainder of the culture. Fold growth and TNC that are shown, arefrom a scaled experiment that started with 30×10̂6 cells. The currentlyused IL-2/IL-15 combination showed the best expansion, but expansion wasalso seen from the cultures pulsed with IL-2 and switched to IL-7/IL-21.Fold expansion was similar in the pulsed conditions to PD0047.

For PD0055, the same cytokine conditions were tested as those assessedfor PD0051 but with CD4+ donor cells (same donor PD0038 as above).Cytokine conditions were IL-7 (5 ng/mL) and IL-15 (0.5 ng/mL) for theexperiments. Pulse conditions were an IL-2 pulse for 5 days followed byIL-7 (5 ng/mL), IL-21 (5 ng/mL), throughout the remainder of theculture. Fold growth and TNC are from a scaled experiment that startedwith 30×10̂6 cells. The currently used IL-7/IL-15 combination showed theusual expansion, however better expansion was seen from the culturespulsed with IL-2 and switched to IL-7/IL-21. Panel A shows PD0051 at day15 (D15) and the comparison of growth with various cytokines and thepercent positive cells (EGFRt+, EGFRt+/CD4+, EGFRt+/CD8+, CD3+,CD3+/CD4+, CD3+/CD4+/CD8+, CD3+/CD8+, CD62L+, CD3+/CD8+, CD62L+,CD3+/CD28+, CD27+, CD127+, CD45RA+/CD45RO−,CD45RA+/CD45RO_/CD45RO_/CD45RA−, CD62+/CD28+) in a donor versus donorcytokine comparison. Panel B shows PD0055 at day 17 (D17) and thecomparison of growth with various cytokines and the percent positivecells. Panel C shows the PD0051 growth curve and the comparison ofgrowth with various cytokines, in which the total cell number isexamined after 20 days of growth in culture. Panel D shows PD0051 andthe comparison of growth with various cytokines, as indicated the CD3+cell growth is examined after 20 days of culture growth. Panels C and Dshow a growth curves (total cells and CD3+) for PD0051, comparing growthfollowing incubation of the cells with the indicated various cytokines,through 20 days in culture. Panel E and F shows growth curves (totalcells and CD3+ respectively) for donor PD0055 following incubation withthe indicated various cytokines, at the indicated time point at day 17of growth in culture.

Shown in FIG. 25, is a cytokine testing experiment in which differentcombinations of cytokines were evaluated for their influence on cellexpansion. For sample PD0059, the same cytokine testing experiment asshown for PD0051 was performed except that this experiment involvedtesting a new CD4+ and CD8+ donor (PD0057). Testing the “normal”cytokine conditions using IL-7 (5 ng/mL) and IL-15 (0.5 ng/mL) for CD4'sand IL-2 (50 U/mL) and IL-15 (0.5 ng/mL) for CD8+ cells were performed.Pulse conditions were an IL-2 pulse for 5 days followed by IL-7 (5ng/mL), IL-21 (5 ng/mL), throughout the remainder of the culture. Foldgrowth and TNC are from a scaled experiment that started with 30×10⁶cells. The “normal” cytokine combinations in this study resulted inexpansion; good expansion was seen following pulsing with IL-2 andswitching to IL-7/IL-21.

For PD0078, a similar cytokine study was performed as in PD0051, exceptthat the experiment was performed with a different CD4+ and CD8+ donor(PD0063). The test was performed using “normal” cytokine conditionsusing IL-7 (5 ng/mL) and IL-15 (0.5 ng/mL) for CD4's and IL-2 (50 U/mL)and IL-15 (0.5 ng/mL) for CD8's versus an IL-2 only condition. Allgrowth through day 14 (D+14) was on a normal plane, cultures were thenEGFRt selected. Recovery of viability and growth was not seen followingselection for the CD8+ cells. As shown in Panel A, are the percent ofpositive cells after thawing the cells. Panel B shows the PD0059 PLAT-02cytokine comparison. Panel C shows PD0059 PLAT-02 cytokine comparisonand the CD3+ cell growth. Panel D shows the PD0078 growth curves. PanelD shows the PD0078 growth curves and the CD3+ cell growth.

Shown in FIG. 26, are two flow charts illustrating an initial expansionmethodology and the updated (or “current”) expansion methodology of cellexpansion based on experimentation, which was then used for futureexperimentations.

Shown in FIG. 27, are growth curves for samples from PD0080, PD0084, andPD0085 and a comparison between all three. These three experiments wererepeated experiments of the “early expansion” methodology. For theseexperiments, the standard cytokine conditions were used: IL-2 (50U/mL)+IL-15 (0.5 ng/mL) for CD8+ T-cells and IL-7 (5 ng/mL)+IL-15 (0.5ng/mL) for CD4+ T-cells. For PD0080, two starting densities were testedin duplicate using PD0063 CD8+ T-cells. One of each of the cellduplicates received volumes of up to 0.7×10⁶ cell/mL following a 3-hourincubation on transduction day. For PD0084, two additional CD8+ donorT-cells were assessed for effects of early expansion. 30×10⁶ cellstarting conditions were used as a control for each donor and a 60×10⁶cell starting density was used with early expansion as test conditions.For PD0085, a similar test to PD0084 was performed except the CD4+T-cells were used to test early expansion. Again 30×10⁶ starting celldensity were used as “normal” control condition and 60×10⁶ cell startingdensity was used as a test condition.

As shown in FIG. 28, the starting material of PD0038 included selectedCD4+ and CD8+ T-cells. The growth curves show TNC from both V-197 bagsof cells that were pooled together. From the early development, it wasintended to pool to 50:50 CD4:CD8 product prior to cryopreservation.Flow data shows post thaw of the pooled product. Bead removal and EGFRtenrichment occurred D+14 for CD4+ and D+15 for CD8+ T-cells. CD8+T-cells were grown in IL-2 (50 U/mL)/IL-15 (0.5 ng/mL) and CD4+ T-cellswere grown in IL-7 (5 ng/mL)/IL-15 (0.5 ng/mL). This process was changedto a cryopreservation of separate products and infusing a 50:50 productupon thawing.

Shown in FIG. 29, is the growth curve for PD0046. Growth curves show TNCfrom both V-197 bags of cells together. In early development there wasan intent to pool a 50:50 CD4:CD8 product prior to cryopreservation.Flow data shows post thaw of the pooled product. Bead removal and EGFRtenrichment then occurred. D+14 for CD4+ T-cells and D+15 for CD8+T-cells. CD8+ T-cells were grown in IL-2 (50 U/mL)/IL-15 (0.5 ng/mL) andCD4+ T-cells were grown in IL-7 (5 ng/mL)/IL-15 (0.5 ng/mL). However,not much was done following this product, due to the donor cells thawingpoorly. Cells were used later to test 10% HA thawing and proved to bemuch better.

Shown in FIG. 30, is the growth curve for PD0063. Growth curves show TNCfrom both V-197 bags of cells together. Bead removal and EGFRtenrichment then occurred D+14 for CD4's and D+15 for CD8's are shown.CD8+ cells were grown in IL-2 (50 U/mL)/IL-15 (0.5 ng/mL) and CD4+ cellswere grown in IL-7 (5 ng/mL)/IL-15 (0.5 ng/mL). Bead removal and EGFRtenrichment then occurred.

Shown in FIG. 31, is the expansion of cells from bulk PBMC cultures whengrown in the presence of cytokines. As shown, CD4+ cells () were grownin the presence of IL2 and IL15. CD8+ cells (▪) were grown in thepresence of IL7 and IL15. The lower panel, (phenotypic comparison) ofFIGS. 31A and 31B, shows percentage of total cells expressing theindicated markers on their surfaces following incubation with thedifferent conditions, each of which resulted in cells expressing markersof engraftment fitness, e.g., CD62L, CD28, CD27, CD45RA. GrowingCD8-enriched cultures in IL-2/IL-15 and CD4-enriched cultures inIL-7/IL-15 yielded robust expansion of both cultures and strongexpression of memory phenotype.

Shown in FIG. 32, is the expansion of enriched CD8+ and CD4+ cells incytokine mixtures. As shown in sample PD0044, enriched CD8+ T-cells wereexpanded in the presence of IL2 and IL15 (top panel). In sample PD0044,enriched CD4+ T-cells were expanded in the presence of IL7 and IL15 forover 20 days. As shown, the pooled CD4+ and CD8+ T-cells expressed CD3,CD4, CD8, CD62L, CD28, CD27, CD45RA and CD45RO. Also tested was sample14602-S14 (See FIG. 32B). As shown, growing CD8+ enriched cultures inIL-2/IL-15 and CD4+ enriched cultures in IL-7/IL-15 yielded robustexpansion of both cultures and strong expression of memory phenotype.Importantly, from the data generated and shown in FIGS. 31 and 32, CD4+T-cells and CD8+ T-cells grew optimally with different cytokinecombinations such that CD4+ T-cells performed best in these studies withIL7 and IL15, while CD8+ T-cells grew best in these studies with thecytokines IL2 and IL15. Growth with their respective optimal cytokinecombinations led to cells that had a phenotype following cell expansionin which the cells expressed the surface markers CD45RA, CD62L, CD28,and CD27, which indicate a high or improved or enhanced engraftmentfitness upon adoptive transfer.

Shown in FIG. 33, are samples from 14602-S01 through 14602-S06 using theoriginal PLAT-02 (Phase I and Phase II of clinical trials) methodologyof cell expansion. The cells were positive for EGFRt prior tocyropreservation. The cells were tested for viability. As shown in thebottom table, the cells 14602-S03 CD8+ and 14602-S03-CD8+ cells did notexpress CD3, CD62L, CD27, CD127, CD45RA/CD45RO− or CD45RA. Shown in FIG.34, are tests on the cell product 14602-SO4 and 14602-SO4-02 which haveboth CD4+ and CD8+ T-cells. The cells were grown in cultures using the“early expansion” methodology. The cells were tested positive for EGFRtexpression prior to cryopreservation.

Shown in FIG. 35, are the cells from samples 14602-S07, 14602-S08, and14602-S09. The CD4+ and CD8+ cells were expanded using the earlyexpansion methodology. In the top panel, as shown in the table for14602-S07 CD4+, cells and 14602-S07 CD8+ cells, shows percentage ofcells expressing various markers including CD3, CD62L, CD27, CD27,CD127, and CD45RA+ at various times. In the bottom panel for sample14602-S08, CD4+ and CD8+ enriched T-cells remained viable after 10 daysin culture.

Shown in FIG. 36, are the cells tested for viability from the batchsamples of 14602-S10, 14602-S11, 14602-S12 and 14602-S13. As shown inthe accompanying tables, the cells all expressed EGFRt prior tocryopreservation. The cells were tested for viability in cell culturefor at least 12 days.

Shown in FIG. 37, are the cells tested for viability from the batchsamples of 14602-S14, 14602-S15 and 14602-S16. As shown in theaccompanying tables, the cells all expressed EGFRt prior tocryopreservation. The cells were tested for viability in cell culturefor at least 12 days.

As shown in FIG. 38, the cells from the sample of 14602, which CD4+ andCD8+ T-cells were enriched from, were positive for CD3, CD62L, CD28,CD127, CD45RA+/CD45RO−, CD45RA and CD45RO. These are specific markerswhich to indicate that both CD4+ and CD8+ cells had a high level ofengraftment fitness following the treatment with their specific cytokinecombinations.

Shown in FIG. 39, are the results for a mouse after T-celladministration after tumor inoculation (survival, tumor progression). Asshown, the mouse injected with the cells from the sample PD0046indicated that the cells had a high, improved, or enhanced engraftmentfitness due to the survival of the mouse after 80 days of administrationusing multiple concentrations of cells. The sample PD00044 did not havea high, improved, or enhanced engraftment fitness as the percentsurvival dropped at day 40 (FIG. 39B).

As shown in FIG. 40 is the average tumor progression in mice treatedwith cells from the PD0044 and PD0046 cell batches. As shown in thegraphs, the tumor progression in the mouse decreased if they wereadministered the CD8+ and CD4+ T-cells from the PD0046 batch, in whichthe cells were originally grown in the appropriate cytokine mixtures forCD4+ and CD8+ T-cells (IL7/IL15 for CD4+ T-cells and IL2/IL15 for CD8+T-cells). As a control, the vehicle cells did not halt tumor progressionin either study with PD0044 and PD0046. As shown in the bottom panels,tumor progression increased in mice treated with cells from the PD0044sample, and in particular increased substantially when treated with theleast concentration of T-cells. Tumor progression decreased incomparison to the vehicle control.

As a further test, three groups of mice were tested in which each grouphad a total number of 5 mice per group. For Group A, the mice weretreated with a placebo of phosphate buffered saline (PBS). Group Butilized cells from “normal” expansion” which had a 1:1 EGFRt+CD4:CD8transduced with Zrx-014. The cells used were from group PD0055 #1 (DonorPD0038 CD4 with 5 ng/ml IL7 & 0.5 ng/ml IL15, S1D14 at 75.9% EGFRt+) andgroup PD0051 #1 (Donor PD0038 CD8 with 50 U/ml IL2 & 0.5 ng/ml IL15,S1D21 @ 76.8% EGFRt+) Group C utilized cells that were “pulsed” withcytokine treatments which comprised 1:1 EGFRt+CD4:CD8 transduced withZrx-014 PD0055 #2 (Donor PD0038 CD4 with 50 U/ml IL2 pulse 5 days, then5 ng/ml IL7 & 5 ng/ml IL21, S1D11 at 81.2% EGFRt+) PD0051 #2 (DonorPD0038 CD8 with 50 U/ml IL2 pulse 5 days, then 5 ng/ml IL7 & 5 ng/mlIL21, S1D21 at 89.5% EGFRt+).

As shown in FIG. 42, are the FACS analysis on cells post thaw that wereused for injection into mice in JME13-25 (PD0051 and PD00055 cells). Asshown both samples of cells had CD8+ and CD4+ T-cells that expressedEGFRt.

As shown in FIG. 43, are the three groups of mice from groups A, B, andC that were treated with placebo PBS (group A), group B (PD00051 “normalexpansion cells”) and group C (PD00055, cells pulsed with cytokinecombinations). As shown, mice that were treated with the placebo inGroup A had tumor progression that is visibly seen at day 20. For theGroup B mice treated with PD0051 (normal expansion cells), the mice, itis shown that tumor progression increases in two mice substantially. Inthe Group C, mice, the mice treated with PD00055 show only a smallamount of tumor progression, therefore indicating the cells treated withthe cytokine mixture prior to use in treatment had a higher engraftmentfitness.

As shown in FIG. 44, is the persistence of the CAR expressing T-cells inthe mice after three days of administration. As indicated, the CD4+T-cells expressing the CARs had a higher persistence compared to thecells of normal expansion as well as the CD8+ T-cells.

Following experiments examining tumor progression after T-celltreatments with CAR expressing T-cells, experiments were performed todetermine if there is an in vivo difference in killing ability betweencells that have been grown in various cytokine conditions in vitro withrepeat antigen encounters (FIG. 45). For the experiment 3 groups of micewere used in which the groups utilized 3 mice each. For Group A, themice were treated with a placebo (PBS). Group B were treated with cellsthat were expanded by the “normal expansion method” (“Normal” 1:1 EGFRt+CD4:CD8 transduced with Zrx-014 PD0055 #1 (Donor PD0038 CD4 with 5 ng/mlIL7 & 0.5 ng/ml IL15, S1D14 @ 75.9% EGFRt+) PD0051 #1 (Donor PD0038 CD8with 50 U/ml IL2 & 0.5 ng/ml IL15, S1D21 @ 76.8% EGFRt+) (FIG. 18).Group C were treated with cells pulsed with cytokines during expansion(“Pulsed” 1:1 EGFRt+CD4:CD8 transduced with Zrx-014 PD0055 #2 (DonorPD0038 CD4 with 50 U/ml IL2 pulse 5 days, then 5 ng/ml IL7 & 5 ng/mlIL21, S1D11 at 81.2% EGFRt+) PD0051 #2 (Donor PD0038 CD8 with 50 U/mlIL2 pulse 5 days, then 5 ng/ml IL7 & 5 ng/ml IL21, S1D21 at 89.5%EGFRt+).

As shown in FIG. 46, group A mice died by day 20 following injectionwith PBS. Group B mice treated with cells that were expanded by the“normal expansion method” had tumor progression in one mouse by day 120.However, for mice treated with the cells that were expanded incytokines, the mice were tumor free by day 120. As such the dataindicate that the cells treated in the cytokine mixtures specific forCD4+ and CD8+ led to a higher engraftment rate and increased survival inthe mice. Additionally, the cells were more efficient in preventingtumor progression.

As shown in FIG. 47, the mice had a 100% survival rate when treated withPD0051 and PD0055 cells post tumor inoculation. However, the tumorprogression was shown to decrease with the treatment using PD0055 cells.

As shown in FIG. 48, the average tumor progression was examined in micetreated with placebo, T-cells grown under “normal expansion” methods,and T-cells that were pulsed with cytokine combinations. For theexperiment at day 0, mice were tumor inoculated and imaged for tumors atday 6. At day 7, T-cells or a placebo was then administered. Tumorinoculation was then repeated at day 14 and day 21.

The mice were examined for tumor progression at day 28, 35, 49, 63, 78,92, 105 and 120, in which images of the tumors were taken. As shown inthe groups of mice, tumor progression decreased in the mice treated withthe cells that were pulsed with cytokines thus indicating that the cellspulsed with cytokines had a higher, improved, or enhanced engraftmentfitness than the cells that were expanded under normal conditionswithout cytokines.

As shown in FIG. 49, experiments were set up to determine if there is anin vivo difference in killing ability between cells that have been grownunder the same conditions as PLAT-01 and PLAT-02 as well as “in between”protocols. For the experiment 17 groups of 5 mice were used test thecells in the treatment of mice after tumor inoculation. The groups weregiven a dose of cells (1.25×10⁶, 2.5×10⁶, 5×10⁶, 10×10⁶) per productcondition as indicated on the Table of FIG. 49. The cells were thawed,counted and injected into the mice on the same day. As shown in FIG. 50,are the amounts of CD4+ and CD8+ cells in the different cell productsfrom the two expansion methods and their amounts of EGFRt in the cells.

As shown in FIG. 51, the 17 groups of mice were tumor inoculated thentreated with dose titrations with T-cells that were expanded undernormal expansion methods or pulsed with cytokine combinations. As seenin the graphs, mice that were dosed with PLAT-1.00 cells at the higherconcentrations had a decrease in tumor progression. Mice that were dosedwith PLAT-2.00 also saw a decrease in tumor progression after 25 days ofadministration at a higher concentration of cells. However, for micedosed with PLAT-1.33, the mice saw an increase in tumor progressionregardless of cell concentration indicating a low or reduced engraftmentfitness for these cells. For mice that were given cells from PLAT-1.67,the mice had a decrease in tumor progression when treated with thehighest concentration of cells in the administration. However as notedin FIG. 50, the cells of PLAT-1.33 also had a low EGFRt count whichcould also indicate a decreased amount of CAR on the cell surface.

Shown in the FIG. 51, is the initiation comparisons between the PD104and the PD116 cell product. For the PD0104 produce, PD0063 CD8+ and CD4+enriched cells were used to repeat experiment to test the expansion ofcells on Tx day to 0.7×10⁶ cell/mL (“early expansion”). For normalPLAT-02 culture conditions, IL-2 (50 U/ml)+IL-15 (0.5 ng/mL) for CD8+cells and IL-7 (5 ng/mL)+IL-15 (0.5 ng/mL) for CD4+ T cells were used.Growth and viability were increased dramatically. These curves mimicwhat was seen in clinical products as far as timing of growth/beadremoval/selection/cryo.

As shown in FIG. 52, mice were treated with dose titrations of cellproducts of PLAT-1.00, PLAT-1.33, PLAT-1.67 and PLAT-2.00. As shown inall graphs, the lowest concentration of cells was least effective in thetreatment of tumor progression. However, the cells that showed the mostengraftment fitness were the PLAT-1.00 and the PLAT-2.00 at higherconcentration.

As shown in FIG. 53, all the cells were least effective at concentration1.25×10⁶. The cells from PLAT-1.00 showed to be the most effective atinhibiting tumor progression indicating that the engraftment fitness ofthe cells were superior or improved, or enhanced as compared to theother groups and indicated that the treatment with cytokines alsopromoted, improved, or enhanced engraftment fitness for the cells. Asshown at a concentration of 5×10⁶, the tumor progression was halted at25 days in comparison to the other groups of cells used to treat themice.

As shown in FIG. 54, the mice had a higher rate of survival if treatedwith a dose of cells between 2.5×10⁶ and 10×10⁶ cells per dose. However,the mice treated with the cells from the PLAT-1.00 product had a longersurvival rate indicating that the T-cells from this group were morerobust and exhibited a higher, improved, or enhanced engraftmentfitness.

As shown in FIG. 55, the survival of the mice was dependent on the doseconcentration of the cells. For all cells, the survival of the micedecreased with the lowest dose of T-cell treatments. However, aspreviously shown the mice treated with the cells from the PLAT-1.00product had a longer survival rate indicating that the T-cells from thisgroup were more robust and had an improved engraftment fitness.

Additional Alternatives

In some alternatives, a method of making genetically modified T-cells,which have a chimeric antigen receptor is provided, wherein the methodcomprises separating, isolating, or enriching a CD8+ population ofT-cells and/or a CD4+ population of T-cells from a mixed population ofT-cells, such as T-cells that are derived from thymocytes or T-cellsthat are derived from engineered precursors, desirably iPS cells, so asto generate an isolated population of T-cells, stimulating the obtainedpopulation of T-cells so as to generate a stimulated population of CD8+T-cells and/or CD4+ T-cells, transducing the stimulated population ofCD8+ T-cells and/or CD4+ T-cells with a vector, wherein the vectorencodes a chimeric antigen receptor and a marker sequence, wherein saidmarker sequence encodes a cell surface selectable marker, so as togenerate a transduced population of CD8+ T-cells and/or CD4+ T-cells,contacting the transduced population of CD8+ T-cells and/or CD4+ T-cellswith at least one cytokine so as to generate a transduced,cytokine-stimulated population of CD8+ T-cells and/or CD4+ T-cells,enriching the transduced, cytokine-stimulated population of CD8+ T-cellsand/or CD4+ T-cells by selection of the marker sequence so as togenerate an enriched population of transduced, cytokine-stimulated CD8+T-cells and/or CD4+ T-cells and propagating the enriched population oftransduced, cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells for atleast one day so as to obtain said genetically modified T-cells, whichhave a chimeric antigen receptor.

In some alternatives, the separating of the CD8+ population of T-cellsand/or a CD4+ population of T-cells from a mixed population of T-cellsis performed by affinity selection for T-cells having an epitope presenton CD8 and/or CD4. In some alternatives, the separating of the CD8+population of T-cells and/or a CD4+ population of T-cells from a mixedpopulation of T-cells is performed by flow cytometry. In somealternatives, the separating of the CD8+ population of T-cells and/or aCD4+ population of T-cells from a mixed population of T-cells isperformed by immuno-magnetic selection. In some alternatives, thegenetically modified CD8+ T-cells and/or CD4+ T-cells comprise at leastone receptor that promotes engraftment fitness. In some alternatives,the at least one receptor is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57,CD137, CD27, CD28 and/or CD62L. In some alternatives, the at least onereceptor is CD27, CD28 and/or CD62L. In some alternatives, thestimulating of the isolated population of T-cells is performed bycontacting the CD8+ and/or CD4+ T-cells with an antibody-bound support,such as a bead or particle. In some alternatives, the antibody-boundsupport comprises anti-TCR, anti-CD2, anti-CD3, anti-CD4 and/oranti-CD28 antibodies. In some alternatives, the antibody-bound supportcomprises anti-CD3 and/or anti-CD28 antibodies. In some alternatives,the vector further comprises a first sequence encoding a leadersequence, a second sequence encoding a ligand binding domain, a thirdsequence encoding a signaling domain and a fourth sequence encoding aselectable marker sequence. In some alternatives, the vector furthercomprises a sequence encoding a spacer. In some alternatives, the spacercomprises an IgG4 hinge. In some alternatives, the vector is a viralvector. In some alternatives, the viral vector is derived from simianvirus 40, adenoviruses, adeno-associated virus (AAV), lentivirus, orretroviruses. In some alternatives, the viral vector is a recombinantadenovirus, adeno-associated virus, lentivirus or retrovirus vector. Insome alternatives, the viral vector is a lentivirus vector. In somealternatives, the marker sequence encodes a truncated epidermal growthfactor receptor (EGFRt). In some alternatives, the at least one cytokinecomprises GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2, and/or IL-21. In somealternatives, the at least one cytokine comprises IL/7, IL-15 and/orIL-21. In some alternatives, the at least one cytokine comprises IL-2,IL-15 and/or IL-21. In some alternatives, the contacting is performedfor 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 days or a period of time within a range defined by any two of thesevalues. In some alternatives, the method is performed with isolated CD4+cells in the absence of CD8+ cells. In some alternatives, the method isperformed with isolated CD8+ in the absence of or enriched over CD4+cells. In some alternatives, the CD4+ expressing T-cells are propagatedfor at least 1 day and may be propagated for 20 days, such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days orfor a period that is within a range defined by any two of theaforementioned time periods. In some alternatives, the CD8+ expressingT-cells are propagated for at least 1 day and may be propagated for 20days, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 days or for a period that is within a range defined by anytwo of the aforementioned time periods. In some alternatives, the methodfurther comprises removing the antibody-bound support, such as beads orparticles. In some alternatives, the ligand binding domain of thechimeric antigen receptor comprises an antibody, or a binding portionthereof. In some alternatives, the ligand binding domain of the chimericantigen receptor comprises a single chain variable fragment (scFv), or abinding portion thereof. In some alternatives, the ligand binding domainof the chimeric antigen receptor comprises FMC63, or a binding portionthereof. In some alternatives, the ligand binding domain of the chimericantigen receptor is specific for CD19. In some alternatives, the methodfurther comprises cryopreserving the genetically modified CD8+ and/orCD4+ T-cells.

In some alternatives, a population of genetically modified T-cells isprovided wherein the population of genetically modified T-cellscomprises a plurality of affinity selected CD8+ and/or CD4+ T-cells, inthe absence of, enriched over, or isolated from CD8− and/or CD4−T-cells, wherein said plurality of affinity selected CD8+ and/or CD4+T-cells have stimulated CD2, CD3, CD4 and/or CD28 receptors, whereinsaid plurality of affinity selected CD8+ and/or CD4+ T-cells furthercomprise a gene encoding a chimeric antigen receptor and a cell surfaceselectable marker and, wherein said plurality of affinity selected CD8+and/or CD4+ T-cells have been re-stimulated with at least one cytokine.In some alternatives, the at least one cytokine comprises GM-CSF, IL-7,IL-12, IL-18, IL-15, IL-2, and/or IL-21. In some alternatives, the atleast one cytokine comprises IL/7, IL-15 and/or IL-21. In somealternatives, the at least one cytokine comprises IL-2, IL-15 and/orIL-21. In some alternatives, the said plurality of affinity selectedCD8+ and/or CD4+ T-cells further comprise at least one receptor thatpromotes, induces, contribute to or enhances engraftment fitness. Insome alternatives, the at least one receptor that promotes, induces,contribute to or enhances engraftment fitness is CD45 RA, CD45 RO, CCR7,CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In some alternatives,the at least one receptor that promotes, induces, contribute to orenhances engraftment fitness is CD27, CD28 and/or CD62L. In somealternatives, the plurality of affinity selected CD8+ and/or CD4+T-cells further comprises a vector having a first sequence encoding aleader sequence, a second sequence encoding a ligand binding domain, athird sequence encoding a signaling domain and a fourth sequenceencoding a selectable marker sequence. In some alternatives, the vectorfurther comprises a sequence encoding a spacer. In some alternatives,the spacer comprises an IgG4 hinge. In some alternatives, the vector isa viral vector. In some alternatives, the viral vector is derived fromsimian virus 40, adenoviruses, adeno-associated virus (AAV), lentivirus,or retroviruses. In some alternatives, the viral vector is a recombinantadenovirus, adeno-associated virus, lentivirus or retrovirus vector. Insome alternatives, the viral vector is a lentivirus vector. In somealternatives, the cell surface selectable marker encodes for a truncatedepidermal growth factor receptor (EGFRt). In some alternatives, theligand binding domain comprises an antibody, or a binding portionthereof. In some alternatives, the ligand binding domain comprises asingle chain variable fragment (scFv), or a binding portion thereof. Insome alternatives, the ligand binding domain comprises FMC63, or abinding portion thereof. In some alternatives, the ligand binding domainis specific for CD19. In some alternatives, the population comprisesisolated CD4+ T-cells in the absence of or enriched over CD8+ T-cells.In some alternatives, the population comprises isolated CD8+ T-cells inthe absence of or enriched over CD4+ T-cells.

In some alternatives, a composition or product combination for humantherapy is provided, wherein the composition or product combinationcomprises a pharmaceutical excipient and at least one population ofgenetically modified T-cells. In some alternatives, the at least onepopulation of genetically modified T-cells comprises a plurality ofaffinity selected CD8+ and/or CD4+ T-cells, in the absence of, enrichedover, or isolated from CD8− and/or CD4− T-cells, wherein said pluralityof affinity selected CD8+ and/or CD4+ T-cells have stimulated CD2, CD3,CD4 and/or CD28 receptors, wherein said plurality of affinity selectedCD8+ and/or CD4+ T-cells further comprise a gene encoding a chimericantigen receptor and a cell surface selectable marker and, wherein saidplurality of affinity selected CD8+ and/or CD4+ T-cells have beenre-stimulated with at least one cytokine. In some alternatives, the atleast one cytokine comprises GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-2,and/or IL-21. In some alternatives, the at least one cytokine comprisesIL/7, IL-15 and/or IL-21. In some alternatives, the at least onecytokine comprises IL-2, IL-15 and/or IL-21. In some alternatives, thesaid plurality of affinity selected CD8+ and/or CD4+ T-cells furthercomprise at least one receptor that promotes, induces, contribute to orenhances engraftment fitness. In some alternatives, the at least onereceptor that promotes, induces, contribute to or enhances engraftmentfitness is CD45 RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28and/or CD62L. In some alternatives, the at least one receptor thatpromotes, induces, contribute to or enhances engraftment fitness isCD27, CD28 and/or CD62L. In some alternatives, the plurality of affinityselected CD8+ and/or CD4+ T-cells further comprises a vector having afirst sequence encoding a leader sequence, a second sequence encoding aligand binding domain, a third sequence encoding a signaling domain anda fourth sequence encoding a selectable marker sequence. In somealternatives, the vector further comprises a sequence encoding a spacer.In some alternatives, the spacer comprises an IgG4 hinge. In somealternatives, the vector is a viral vector. In some alternatives, theviral vector is derived from simian virus 40, adenoviruses,adeno-associated virus (AAV), lentivirus, or retroviruses. In somealternatives, the viral vector is a recombinant adenovirus,adeno-associated virus, lentivirus or retrovirus vector. In somealternatives, the viral vector is a lentivirus vector. In somealternatives, the cell surface selectable marker encodes for a truncatedepidermal growth factor receptor (EGFRt). In some alternatives, theligand binding domain comprises an antibody, or a binding portionthereof. In some alternatives, the ligand binding domain comprises asingle chain variable fragment (scFv), or a binding portion thereof. Insome alternatives, the ligand binding domain comprises FMC63, or abinding portion thereof. In some alternatives, the ligand binding domainis specific for CD19. In some alternatives, at least one populationcomprises isolated CD4+ T-cells in the absence of or enriched over CD8+T-cells. In some alternatives, at least one population comprisesisolated CD8+ T-cells in the absence of or enriched over CD4+ T-cells.In some alternatives, the composition or product combination comprisesthe population of genetically modified T-cells, wherein the populationof genetically modified T-cells comprises isolated CD8+ T-cells in theabsence of or enriched over CD4+ T-cells. In some alternatives, thecomposition or product combination comprises the population ofgenetically modified T-cells, wherein the population of geneticallymodified T-cells comprises isolated CD4+ T-cells in the absence of orenriched over CD8+ T-cells. In some alternatives, the populationcomprises isolated CD8+ T-cells in the absence of or enriched over CD4+T-cells and isolated CD4+ T-cells in the absence of or enriched overCD8+ T-cells mixed or co-administered in a 1:1 ratio.

In some alternatives, a method of treating, inhibiting, or amelioratinga disease in a subject in need thereof is provided, wherein the methodcomprises administering to the subject at least one composition orproduct combination. In some alternatives, the at least one compositionor product combination comprises a pharmaceutical excipient and at leastone population of genetically modified T-cells. In some alternatives,the at least one population of genetically modified T-cells comprises aplurality of affinity selected CD8+ and/or CD4+ T-cells, in the absenceof, enriched over or isolated from CD8− and/or CD4− T-cells, whereinsaid plurality of affinity selected CD8+ and/or CD4+ T-cells havestimulated CD2, CD3, CD4 and/or CD28 receptors, wherein said pluralityof affinity selected CD8+ and/or CD4+ T-cells further comprise a geneencoding a chimeric antigen receptor and a cell surface selectablemarker and, wherein said plurality of affinity selected CD8+ and/or CD4+T-cells have been re-stimulated with at least one cytokine. In somealternatives, the at least one cytokine comprises GM-CSF, IL-7, IL-12,IL-15, IL-18, IL-2, and/or IL-21. In some alternatives, the at least onecytokine comprises IL/7, IL-15 and/or IL-21. In some alternatives, theat least one cytokine comprises IL-2, IL-15 and/or IL-21. In somealternatives, the said plurality of affinity selected CD8+ and/or CD4+T-cells further comprise at least one receptor that promotes engraftmentfitness. In some alternatives, the at least one receptor that promotes,induces, contribute to or enhances engraftment fitness is CD45 RA, CD45RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. In somealternatives, the at least one receptor that promotes, induces,contribute to or enhances engraftment fitness is CD27, CD28 and/orCD62L. In some alternatives, the plurality of affinity selected CD8+and/or CD4+ T-cells further comprises a vector having a first sequenceencoding a leader sequence, a second sequence encoding a ligand bindingdomain, a third sequence encoding a signaling domain and a fourthsequence encoding a selectable marker sequence. In some alternatives,the vector further comprises a sequence encoding a spacer. In somealternatives, the spacer comprises an IgG4 hinge. In some alternatives,the vector is a viral vector. In some alternatives, the viral vector isderived from simian virus 40, adenoviruses, adeno-associated virus(AAV), lentivirus, or retroviruses. In some alternatives, the viralvector is a recombinant adenovirus, adeno-associated virus, lentivirusor retrovirus vector. In some alternatives, the viral vector is alentivirus vector. In some alternatives, the cell surface selectablemarker encodes for a truncated epidermal growth factor receptor (EGFRt).In some alternatives, the ligand binding domain comprises an antibody,or a binding portion thereof. In some alternatives, the ligand bindingdomain comprises a single chain variable fragment (scFv), or a bindingportion thereof. In some alternatives, the ligand binding domaincomprises FMC63, or a binding portion thereof. In some alternatives, theligand binding domain is specific for CD19. In some alternatives, atleast one population comprises isolated CD4+ T-cells in the absence ofor enriched over CD8+ T-cells. In some alternatives, at least onepopulation comprises isolated CD8+ T-cells in the absence of or enrichedover CD4+ T-cells. In some alternatives, the composition or productcombination comprises the population of genetically modified T-cells,wherein the population of genetically modified T-cells comprisesisolated CD8+ T-cells in the absence of or enriched over CD4+ T-cells.In some alternatives, the composition or product combination comprisesthe population of genetically modified T-cells, wherein the populationof genetically modified T-cells comprises isolated CD4+ T-cells in theabsence of or enriched over CD8+ T-cells. In some alternatives, thepopulation comprises isolated CD8+ T-cells in the absence of or enrichedover CD4+ T-cells and isolated CD4+ T-cells in the absence of orenriched over CD8+ T-cells mixed or co-administered in a 1:1 ratio. Insome alternatives, the method comprises administering the composition orproduct combination, wherein the composition or product combinationcomprises at least one population, wherein the at least one populationcomprises isolated CD4+ T-cells in the absence of or enriched over CD8+T-cells. In some alternatives, the method comprises administering thecomposition or product combination, wherein the composition or productcombination comprises at least one population, wherein the at least onepopulation comprises isolated CD8+ T-cells in the absence of or enrichedover CD4+ T-cells. In some alternatives, the method comprisesadministering the composition or product combination, wherein thecomposition or product combination comprises at least one population,wherein the at least one population comprises isolated CD4+ T-cells inthe absence of or enriched over CD8+ T-cells and isolated CD8+ T-cellsin the absence of or enriched over CD4+ T-cells mixed or co-administeredin a 1:1 ratio. In some alternatives, the method further comprisesadministering the composition or product combination, wherein thecomposition or product combination comprises at least one population,wherein the at least one population comprises isolated CD8+ T-cells inthe absence of or enriched over CD4+ T-cells. In some alternatives, themethod further comprises administering the composition or productcombination, wherein the composition or product combination comprises atleast one population, wherein the at least one population comprisesisolated CD4+ T-cells in the absence of or enriched over CD8+ T-cells.In some alternatives, the subject is identified or selected to receivean anti-cancer therapy. In some alternatives, the method furthercomprises measuring or evaluating an inhibition of a disease. In somealternatives, the method further comprises providing said subject anadditional anti-cancer therapy before, during, or after administrationof the composition or product combination. In some alternatives, thecomposition or product combinations are administered to said subject byadoptive cell transfer. In some alternatives, the composition or productcombinations are administered to said subject after said subject hasreceived another form of anti-cancer therapy. In some alternatives, thecomposition or product combinations are administered to said subjectafter said subject has received another form of anti-cancer therapy. Insome alternatives, the subject is suffering from leukemia. In somealternatives, the subject has recurrent and/or chemotherapy refractoryCD19+ childhood acute lymphoblastic leukemia (ALL). In somealternatives, the subject has recurrent and/or chemotherapy refractoryCD19+ acute lymphoblastic leukemia (ALL). In some alternatives, thesubject is suffering from an autoimmune disease. In some alternatives,the subject is suffering from a post-HSCT relapse.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to alternatives containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

More Alternatives

In some alternatives, the separating, enriching, or isolating of theCD8+ expressing population of T-cells and/or a CD4+ expressingpopulation of T-cells, such as T-cells that are derived from thymocytesor T-cells that are derived from engineered precursors, desirably iPScells, from a mixed population of T-cells is performed by affinityselection for T-cells having an epitope present on CD8 and/or CD4. Insome alternatives, the separating, enriching, or isolating of the CD8+expressing population of T-cells and/or a CD4+ expressing population ofT-cells from a mixed population of T-cells is performed by flowcytometry. In some alternatives, the separating, enriching, or isolatingof the CD8+ expressing population of T-cells and/or a CD4+ expressingpopulation of T-cells from a mixed population of T-cells is performed byimmuno-magnetic selection. In some alternatives, the geneticallymodified CD8+ expressing T-cells and/or CD4+ expressing T-cells compriseat least one receptor that promotes, induces, or contributes toengraftment fitness. In some alternatives, the at least one receptorthat promotes, induces, or contributes to engraftment fitness is CD45RA, CD45 RO, CCR7, CD25, CD127, CD57, CD137, CD27, CD28 and/or CD62L. Inpreferred alternatives, the at least one receptor that promotes,induces, or contributes to engraftment fitness is CD27, CD28 and/orCD62L. In some alternatives, the stimulating of the isolated, enriched,or separated population of T-cells is performed by contacting the CD8+and/or CD4+ expressing T-cells with an antibody-bound support, such as abead or particle. In some of these alternatives, the antibody-boundsupport comprises anti-TCR, anti-CD2, anti-CD3, anti-CD4 and/oranti-CD28 antibodies. In preferred alternatives, the antibody-boundsupport comprises anti-CD3 and/or anti-CD28 antibodies.

In many of the aforementioned alternatives, the vector further comprisesa first sequence encoding a leader sequence, a second sequence encodinga ligand binding domain, a third sequence encoding a signaling domainand a fourth sequence encoding a selectable marker sequence. In some ofthese alternatives, the vector further comprises a sequence encoding aspacer, which in some alternatives may comprise an IgG4 hinge. In manyof the aforementioned alternatives, the vector is a viral vector or amini-circle.

In many of the aforementioned alternatives, the viral vector is derivedfrom simian virus 40, adenoviruses, adeno-associated virus (AAV),lentivirus, or retroviruses. In some alternatives, the viral vector is arecombinant adenovirus, adeno-associated virus, lentivirus or retrovirusvector. Preferably, the viral vector is a lentivirus vector. In many ofthe aforementioned alternatives, the marker sequence encodes a truncatedepidermal growth factor receptor (EGFRt). In many of the aforementionedalternatives, the at least one cytokine that is utilized comprisesGM-CSF, IL-7, IL-12, IL-15, IL-18, IL-15, IL-2, and/or IL-21 and saidcytokine is provided exogenously to the T-cells e.g., in addition to anycytokine that may be produced by the cells or present in media.

In desirable alternatives, the at least one cytokine comprises IL-7,IL-15 and/or IL-21. In many of the aforementioned alternatives, the atleast one cytokine comprises IL-2, IL-15 and/or IL-21. In desirablealternatives, the at least one cytokine comprises IL-21 and anothercytokine, and/or comprises IL-7 and at least one other cytokine, and/orcomprises IL-15 and at least one other cytokine, such as comprisingIL-21 and IL-15, comprising IL-21 and IL-7, or comprising IL-15 andIL-7, or comprising IL-2 and IL-15, or comprising IL-7 and IL-15. Inpreferred alternatives, the contacting period is performed for at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20days or a period of time within a range defined by any two of these timepoints. In many of the aforementioned alternatives, the methods areperformed with isolated, separated, or enriched populations of CD4+expressing T-cells, such as T-cells that are derived from thymocytes orT-cells that are derived from engineered precursors, desirably iPScells, in the absence of or having a reduced amount CD8+ expressingT-cells, as compared to a native population of unseparated,non-enriched, or non-isolated population of T-cells. In many of theaforementioned alternatives, these methods are performed with isolated,separated, or enriched populations of CD8+ expressing T-cells, such asT-cells that are derived from thymocytes or T-cells that are derivedfrom engineered precursors, desirably iPS cells, in the absence of orhaving a reduced amount of CD4+ expressing T-cells, as compared to anative population of unseparated, non-enriched, or non-isolatedpopulation of T-cells. In many of the aforementioned alternatives, theCD4+ expressing T-cells are propagated for at least 1 day, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 daysor any time that is within a range of times defined by any two of theaforementioned time points. In many of the aforementioned alternatives,the CD8+ expressing T-cells are propagated for at least 1 day, such as1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20days or any time that is within a range of times defined by any two ofthe aforementioned time points.

In some alternatives, a method of making genetically modified T-cells,which have a chimeric antigen receptor is provided, wherein the methodcomprises separating or enriching a CD8+ expressing population ofT-cells and/or a CD4+ expressing population of T-cells, such as T-cellsthat are derived from thymocytes or T-cells that are derived fromengineered precursors, wherein the precursors are optionally iPS cells,from a mixed population of T-cells, so as to generate a separated orenriched population of T-cells, stimulating the separated or enrichedpopulation of T-cells so as to generate a stimulated population of CD8+T-cells and/or CD4+ T-cells, transducing the stimulated population ofCD8+ T-cells and/or CD4+ T-cells with a vector, wherein the vectorencodes a chimeric antigen receptor so as to generate a transducedpopulation of CD8+ T-cells and/or CD4+ T-cells, contacting thetransduced population of CD8+ T-cells and/or CD4+ T-cells with at leastone cytokine, provided exogenously, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or for a period that iswithin a range defined by any two of the aforementioned time periods, soas to generate a transduced, cytokine-stimulated population of CD8+T-cells and/or CD4+ T-cells, wherein the method thereby obtains saidgenetically modified T-cells, which have a chimeric antigen receptor. Insome alternatives, the method further comprises enriching thetransduced, cytokine-stimulated population of CD8+ T-cells and/or CD4+T-cells by selection of a marker, so as to generate an enrichedpopulation of transduced, cytokine-stimulated CD8+ T-cells and/or CD4+T-cells. In some alternatives, the marker is encoded by the vector andoptionally is a cell surface marker. In some alternatives, the methodfurther comprises further comprising propagating the enriched populationof transduced, cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells forat least one day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 days or for a period that is within a rangedefined by any two of the aforementioned time periods.

In some alternatives, a method of producing genetically modified T cellsis provided, wherein the method comprises introducing a vector encodinga recombinant protein into cells of a CD4+-enriched T cell composition,and incubating cells of the CD4+ enriched T cell composition with acombination of cytokines comprising IL-7 and IL-15, and/or introducing avector encoding a recombinant protein into cells of a CD8+ enriched Tcell composition and incubating cells of the CD8+ enriched T cellcomposition with a combination of cytokines comprising IL-2 and IL-15,wherein the method thereby generates a transduced CD4+ enrichedpopulation and/or a transduced CD8+ enriched population. In somealternatives, the transduced CD4+ enriched population has a higher levelof surface expression of CD62L, CD27, and/or CD28, as compared to areference enriched population of transduced CD4+ cells, and/or has anincreased indicator of engraftment fitness and/or expansion, and/orpersistence, upon administration to a subject as compared to thereference enriched population of transduced CD4+ cells, wherein thereference population is a CD4+-enriched population prepared by anothermethod, which is identical to the method, except that the IL-2 alone isused in place of the combination of cytokines. In some alternatives, thetransduced CD8+ enriched population has a higher level of surfaceexpression of CD62L, CD27, and/or CD28, as compared to a referenceenriched population of transduced CD8+ cells, and/or has an increasedindicator of engraftment fitness and/or expansion, and/or persistence,upon administration to a subject as compared to the reference enrichedpopulation of transduced CD8+ cells, wherein the reference population isa population of CD8+-enriched cells prepared by another method that isidentical to the method, except that the IL-2 alone is used in place ofthe combination of cytokines.

In some alternatives, a method of producing genetically modified T cellsis provided, wherein the method comprises introducing a vector encodinga recombinant protein into cells of a CD4+ enriched T cell compositionand incubating cells of the CD4+ enriched T cell composition with afirst combination of cytokines and introducing a vector encoding arecombinant protein into cells of a CD8+ enriched T cell composition andincubating cells of the CD8+ enriched T cell composition with a secondcombination of cytokines which is distinct from the first combination ofcytokines, wherein the method thereby generates a transduced CD4+enriched population and a transduced CD8+ enriched population. In somealternatives, the transduced CD4+ enriched population has a higher levelof surface expression of CD62L, CD27, and/or CD28, as compared to areference enriched population of transduced CD4+ cells prepared byanother method that is identical to the method, except that the firstand second combination of cytokines are identical, and/or has anincreased indicator of engraftment fitness and/or expansion, and/orpersistence, upon administration to a subject as compared to thereference enriched population of transduced CD4+ cells. In somealternatives, the transduced CD8+ enriched population has a higher levelof surface expression of CD62L, CD27, and/or CD28, as compared to areference enriched population of transduced CD8+ cells prepared byanother method that is identical to the method, except that the firstand second combination of cytokines are identical, and/or has anincreased indicator of engraftment fitness and/or expansion, and/orpersistence, upon administration to a subject as compared to thereference enriched population of transduced CD8+ cells. In somealternatives, the first combination comprises IL-7 and IL-15 and thesecond combination comprises IL-2 and IL-15. In some alternatives, thecytokine combination(s) is added subsequently to transductioninitiation, and optionally on the same day as transduction initiation.In some alternatives, the concentration of IL-2 is at or about 50 U/mL,where applicable, the concentration of IL-15 is at or about 0.5 ng/mL,where applicable, and/or the concentration of IL-7 is at or about 5ng/mL, where applicable. In some alternatives, the addition of thecombination of cytokines to the CD4+-enriched and/or to theCD8+-enriched composition comprises increasing the volume of thecomposition in which the cells are incubated, thereby decreasing celldensity. In some alternatives, the indicator of engraftment fitnesscomprises a percentage of cells in the composition or total surfaceexpression levels for the population of a cell surface marker selectedfrom the group consisting of CD62L, CD27, and/or CD28. In somealternatives, the indicator of engraftment fitness comprises persistencein a subject upon administration. In some alternatives, the cells arederived from the subject. In some alternatives, transduced CD8+ and/orCD4+ cells persist upon administration to a subject from which the cellswere derived, for at least at or about 30 or 60 days post injection ofthe cells into the subject. In some alternatives, the recombinantprotein is a chimeric antigen receptor. In some alternatives, the methodfurther comprises prior to the transduction, enriching a T cellcomposition for cells expressing CD4, thereby generating the enrichedCD4+ enriched T cell composition so-transduced, and/or, prior to thetransduction, enriching a T cell composition for cells expressing CD8,thereby generating the enriched CD8+ enriched T cell compositionso-transduced. In some alternatives, the method further comprisescryopreserving the engineered cells. In some alternatives, the methodfurther comprises administering the engineered cells to a subject, andoptionally further comprising pooling the transduced CD4+ enriched andthe transduced CD8+ enriched compositions prior to said administering.

In some alternatives, a cell or composition is produced by any of thealternatives of the methods listed in the previous paragraphs of thissection of more alternatives. In some alternatives, a method ofadministering the cell produced by any of the alternatives of the methodor the alternatives of the compositions listed in the previousparagraphs is contemplated. In some alternatives, the cell orcomposition is administered to a subject. In some alternatives, theadministration is to a subject from which the cells were derived.

In some alternatives, a method of adoptive cell therapy is provided,wherein the method comprises (a) incubating a CD4+-enriched T cellcomposition under stimulating conditions and in the presence of IL-15and IL-7, thereby generating an expanded CD4+ composition, (b)separately incubating a CD8+-enriched T cell composition understimulating conditions and in the presence of IL-15 and IL-2, therebygenerating an expanded CD8+ composition, and (c) administering theexpanded CD4+ and expanded CD8+ populations to a subject, optionallysimultaneously. In some alternatives, the CD4+ and CD8+ populations areobtained from the subject. In some alternatives, the one or more of therecited cytokines or combinations of cytokines further comprises CD21.In some alternatives, the CD4+-enriched population or the total numberof T cells therein comprises at least 70, 80, 90, 95, 96, 97, 98, or 99%CD4+ cells, and/or the C8+-enriched population or the total number of Tcells therein comprises at least 70, 80, 90, 95, 96, 97, 98, or 99% CD8+cells.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

1. A method of making genetically modified T-cells, which have achimeric antigen receptor, comprising: separating or enriching a CD8+expressing population of T-cells and/or a CD4+ expressing population ofT-cells, such as T-cells that are derived from thymocytes or T-cellsthat are derived from engineered precursors, desirably iPS cells, from amixed population of T-cells so as to generate a separated or enrichedpopulation of T-cells; stimulating the separated or enriched populationof T-cells so as to generate a stimulated population of CD8+ T-cellsand/or CD4+ T-cells; transducing the stimulated population of CD8+T-cells and/or CD4+ T-cells with a vector, wherein the vector encodes achimeric antigen receptor and a marker sequence, wherein said markersequence encodes a cell surface selectable marker, so as to generate atransduced population of CD8+ T-cells and/or CD4+ T-cells; contactingthe transduced population of CD8+ T-cells and/or CD4+ T-cells with atleast one cytokine, which can be provided exogenously to the T-cells,e.g., in addition to any cytokine that may be produced by the cells orpresent in media, such as for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 days or for a period that is within arange defined by any two of the aforementioned time periods, so as togenerate a transduced, cytokine-stimulated population of CD8+ T-cellsand/or CD4+ T-cells; enriching the transduced, cytokine-stimulatedpopulation of CD8+ T-cells and/or CD4+ T-cells by selection of themarker sequence so as to generate an enriched population of transduced,cytokine-stimulated CD8+ T-cells and/or CD4+ T-cells; and propagatingthe enriched population of transduced, cytokine-stimulated CD8+ T-cellsand/or CD4+ T-cells for at least one day, such as 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or for a periodthat is within a range defined by any two of the aforementioned timeperiods, so as to obtain said genetically modified T-cells, which have achimeric antigen receptor. 2.-73. (canceled)